Flynn Lab Publications
Featured Publications
-
2020. Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology. Pubmed: 33052334 DOI:10.1101/2020.10.06.327445 Flynn RA, Belk JA, Qi Y, Yasumoto Y, Schmitz CO, Mumbach MR, Limaye A, Wei J, Alfajaro MM, Parker KR, Chang HY, Horvath TL, Carette JE, Bertozzi C, Wilen CB, Satpathy AT. 2020. Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology. Pubmed: 33052334 DOI:10.1101/2020.10.06.327445 Array -
Shao Z, Flynn RA, Crowe JL, Zhu Y, Liang J, Jiang W, Aryan F, Aoude P, Bertozzi CR, Estes VM, Lee BJ, Bhagat G, Zha S, Calo E. 2020. DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature. 579(7798):291-296. Pubmed: 32103174 DOI:10.1038/s41586-020-2041-2 Shao Z, Flynn RA, Crowe JL, Zhu Y, Liang J, Jiang W, Aryan F, Aoude P, Bertozzi CR, Estes VM, Lee BJ, Bhagat G, Zha S, Calo E. 2020. DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature. 579(7798):291-296. Pubmed: 32103174 DOI:10.1038/s41586-020-2041-2 The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.
All Publications
2024
-
Ducoli L, Zarnegar BJ, Porter DF, Meyers RM, Miao W, Riley NM, Srinivasan S, Jackrazi LV, Yang YY, Li Z, Wang Y, Bertozzi CR, Flynn RA, Khavari PA. 2024. irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA. bioRxiv : the preprint server for biology. Pubmed: 39386644 DOI:10.1101/2024.09.27.615518 Ducoli L, Zarnegar BJ, Porter DF, Meyers RM, Miao W, Riley NM, Srinivasan S, Jackrazi LV, Yang YY, Li Z, Wang Y, Bertozzi CR, Flynn RA, Khavari PA. 2024. irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA. bioRxiv : the preprint server for biology. Pubmed: 39386644 DOI:10.1101/2024.09.27.615518 RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells. -
Zheng R, Dunlap M, Bobkov GOM, Gonzalez-Figueroa C, Patel KJ, Lyu J, Harvey SE, Chan TW, Quinones-Valdez G, Choudhury M, Le Roux CA, Bartels MD, Vuong A, Flynn RA, Chang HY, Van Nostrand EL, Xiao X, Cheng C. 2024. hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing. Molecular cell. 84(11):2087-2103.e8. Pubmed: 38815579 DOI:S1097-2765(24)00397-6 Zheng R, Dunlap M, Bobkov GOM, Gonzalez-Figueroa C, Patel KJ, Lyu J, Harvey SE, Chan TW, Quinones-Valdez G, Choudhury M, Le Roux CA, Bartels MD, Vuong A, Flynn RA, Chang HY, Van Nostrand EL, Xiao X, Cheng C. 2024. hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing. Molecular cell. 84(11):2087-2103.e8. Pubmed: 38815579 DOI:S1097-2765(24)00397-6 RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.Copyright © 2024 Elsevier Inc. All rights reserved. -
Kageler L, Perr J, Flynn RA. 2024. Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology. Cell chemical biology. 31(6):1132-1144. Pubmed: 38772372 DOI:S2451-9456(24)00177-6 Kageler L, Perr J, Flynn RA. 2024. Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology. Cell chemical biology. 31(6):1132-1144. Pubmed: 38772372 DOI:S2451-9456(24)00177-6 Proximity is a fundamental concept in chemistry and biology, referring to the convergence of molecules to facilitate new molecular interactions or reactions. Hybrid biopolymers like glycosylphosphatidylinositol (GPI)-anchored proteins, ubiquitinated proteins, glycosylated RNAs (glycoRNAs), and RNAylated proteins exemplify this by covalent bonding of moieties that are often orthogonally active. Hybrid molecules like glycoRNAs are localized to new physical spaces, generating new interfaces for biological functions. To fully investigate the compositional and spatial features of molecules like glycoRNAs, flexible genetic and chemical tools that encompass different encoding and targeting biopolymers are required. Here we discuss concepts of molecular proximity and explore newer proximity labeling technologies that facilitate applications in RNA biology, cell surface biology, and the interface therein with a particular focus on glycoRNA biology. We review the advantages and disadvantages of methods pertaining to cell surface RNA identification and provide insights into the vast opportunities for method development in this area.Copyright © 2024 Elsevier Ltd. All rights reserved. -
Xie Y, Liu X, Zhao C, Chen S, Wang S, Lin Z, Robison FM, George BM, Flynn RA, Lebrilla CB, Garcia BA. 2024. Development and application of GlycanDIA workflow for glycomic analysis. bioRxiv : the preprint server for biology. Pubmed: 38559279 DOI:10.1101/2024.03.12.584702 Xie Y, Liu X, Zhao C, Chen S, Wang S, Lin Z, Robison FM, George BM, Flynn RA, Lebrilla CB, Garcia BA. 2024. Development and application of GlycanDIA workflow for glycomic analysis. bioRxiv : the preprint server for biology. Pubmed: 38559279 DOI:10.1101/2024.03.12.584702 Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from -glycans, -glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile -glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the -glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation. -
Hristov P, Flynn RA. 2024. Imaging glycosylated RNAs at the subcellular scale. Nature biotechnology. 42(4):574-575. Pubmed: 37872411 DOI:10.1038/s41587-023-02021-1 Hristov P, Flynn RA. 2024. Imaging glycosylated RNAs at the subcellular scale. Nature biotechnology. 42(4):574-575. Pubmed: 37872411 DOI:10.1038/s41587-023-02021-1 -
Peltan EL, Riley NM, Flynn RA, Roberts DS, Bertozzi CR. 2024. Galectin-3 does not interact with RNA directly. Glycobiology. 34(1). Pubmed: 37815932 DOI:10.1093/glycob/cwad076 Peltan EL, Riley NM, Flynn RA, Roberts DS, Bertozzi CR. 2024. Galectin-3 does not interact with RNA directly. Glycobiology. 34(1). Pubmed: 37815932 DOI:10.1093/glycob/cwad076 Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.© The Author(s) 2023. Published by Oxford University Press. -
Xie Y, Chai P, Till NA, Hemberger H, Lebedenko CG, Porat J, Watkins CP, Caldwell RM, George BM, Perr J, Bertozzi CR, Garcia BA, Flynn RA. 2024. The modified RNA base acpU is an attachment site for N-glycans in glycoRNA. Cell. 187(19):5228-5237.e12. Pubmed: 39173631 DOI:S0092-8674(24)00838-9 Xie Y, Chai P, Till NA, Hemberger H, Lebedenko CG, Porat J, Watkins CP, Caldwell RM, George BM, Perr J, Bertozzi CR, Garcia BA, Flynn RA. 2024. The modified RNA base acpU is an attachment site for N-glycans in glycoRNA. Cell. 187(19):5228-5237.e12. Pubmed: 39173631 DOI:S0092-8674(24)00838-9 GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acpU) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.Copyright © 2024 Elsevier Inc. All rights reserved. -
Porat J, Watkins CP, Jin C, Xie X, Tan X, Lebedenko CG, Hemberger H, Shin W, Chai P, Collins JJ, Garcia BA, Bojar D, Flynn RA. 2024. O-glycosylation contributes to mammalian glycoRNA biogenesis. bioRxiv : the preprint server for biology. Pubmed: 39257776 DOI:10.1101/2024.08.28.610074 Porat J, Watkins CP, Jin C, Xie X, Tan X, Lebedenko CG, Hemberger H, Shin W, Chai P, Collins JJ, Garcia BA, Bojar D, Flynn RA. 2024. O-glycosylation contributes to mammalian glycoRNA biogenesis. bioRxiv : the preprint server for biology. Pubmed: 39257776 DOI:10.1101/2024.08.28.610074 There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acpU and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease. 2023
-
Watkins CP, Flynn RA. 2023. A (cross)link in the chains. Nature chemistry. 15(1):5-6. Pubmed: 36609645 DOI:10.1038/s41557-022-01116-7 Watkins CP, Flynn RA. 2023. A (cross)link in the chains. Nature chemistry. 15(1):5-6. Pubmed: 36609645 DOI:10.1038/s41557-022-01116-7 -
Perr J, Langen A, Almahayni K, Nestola GL, Chai P, Lebedenko CG, Volk R, Caldwell RM, Spiekermann M, Hemberger H, Bisaria N, Tzelepis K, Calo E, Möckl L, Zaro B, Flynn RA. 2023. RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry. bioRxiv. DOI:10.1101/2023.09.04.556039 Perr J, Langen A, Almahayni K, Nestola GL, Chai P, Lebedenko CG, Volk R, Caldwell RM, Spiekermann M, Hemberger H, Bisaria N, Tzelepis K, Calo E, Möckl L, Zaro B, Flynn RA. 2023. RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry. bioRxiv. DOI:10.1101/2023.09.04.556039 The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA binding proteins (RBPs) are present on the surface of living cells. These cell surface RBPs (csRBPs) precisely organize into well-defined nanoclusters that are enriched for multiple RBPs, glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell surface interaction for the cell penetrating peptide TAT. Removal of RNA from the cell surface, or loss of RNA binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. -
Leon KE, Khalid MM, Flynn RA, Fontaine KA, Nguyen TT, Kumar GR, Simoneau CR, Tomar S, Jimenez-Morales D, Dunlap M, Kaye J, Shah PS, Finkbeiner S, Krogan NJ, Bertozzi C, Carette JE, Ott M. 2023. Nuclear accumulation of host transcripts during Zika Virus Infection. PLoS pathogens. 19(1):e1011070. Pubmed: 36603024 DOI:10.1371/journal.ppat.1011070 Leon KE, Khalid MM, Flynn RA, Fontaine KA, Nguyen TT, Kumar GR, Simoneau CR, Tomar S, Jimenez-Morales D, Dunlap M, Kaye J, Shah PS, Finkbeiner S, Krogan NJ, Bertozzi C, Carette JE, Ott M. 2023. Nuclear accumulation of host transcripts during Zika Virus Infection. PLoS pathogens. 19(1):e1011070. Pubmed: 36603024 DOI:10.1371/journal.ppat.1011070 Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.Copyright: © 2023 Leon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. -
Miao W, Porter DF, Lopez-Pajares V, Siprashvili Z, Meyers RM, Bai Y, Nguyen DT, Ko LA, Zarnegar BJ, Ferguson ID, Mills MM, Jilly-Rehak CE, Wu CG, Yang YY, Meyers JM, Hong AW, Reynolds DL, Ramanathan M, Tao S, Jiang S, Flynn RA, Wang Y, Nolan GP, Khavari PA. 2023. Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell. 186(1):80-97.e26. Pubmed: 36608661 DOI:S0092-8674(22)01517-3 Miao W, Porter DF, Lopez-Pajares V, Siprashvili Z, Meyers RM, Bai Y, Nguyen DT, Ko LA, Zarnegar BJ, Ferguson ID, Mills MM, Jilly-Rehak CE, Wu CG, Yang YY, Meyers JM, Hong AW, Reynolds DL, Ramanathan M, Tao S, Jiang S, Flynn RA, Wang Y, Nolan GP, Khavari PA. 2023. Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell. 186(1):80-97.e26. Pubmed: 36608661 DOI:S0092-8674(22)01517-3 Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.Published by Elsevier Inc. -
Xie Y, Hemberger H, Till NA, Chai P, Watkins CP, Lebedenko CG, Caldwell RM, George BM, Bertozzi CR, Garcia BA, Flynn RA. 2023. The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA. bioRxiv. DOI:10.1101/2023.11.06.565735 Xie Y, Hemberger H, Till NA, Chai P, Watkins CP, Lebedenko CG, Caldwell RM, George BM, Bertozzi CR, Garcia BA, Flynn RA. 2023. The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA. bioRxiv. DOI:10.1101/2023.11.06.565735 We recently identified glycoRNA—a previously undescribed glycoconjugate—which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. -
Zheng R, Dunlap M, Lyu J, Gonzalez-Figueroa C, Bobkov G, Harvey SE, Chan TW, Quinones-Valdez G, Choudhury M, Vuong A, Flynn RA, Chang HY, Xiao X, Cheng C. 2023. LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity. bioRxiv : the preprint server for biology. Pubmed: 36865202 DOI:10.1101/2023.02.23.529804 Zheng R, Dunlap M, Lyu J, Gonzalez-Figueroa C, Bobkov G, Harvey SE, Chan TW, Quinones-Valdez G, Choudhury M, Vuong A, Flynn RA, Chang HY, Xiao X, Cheng C. 2023. LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity. bioRxiv : the preprint server for biology. Pubmed: 36865202 DOI:10.1101/2023.02.23.529804 RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance. -
Chai P, Lebedenko CG, Flynn RA. 2023. RNA Crossing Membranes: Systems and Mechanisms Contextualizing Extracellular RNA and Cell Surface GlycoRNAs. Annual review of genomics and human genetics. 24:85-107. Pubmed: 37068783 DOI:10.1146/annurev-genom-101722-101224 Chai P, Lebedenko CG, Flynn RA. 2023. RNA Crossing Membranes: Systems and Mechanisms Contextualizing Extracellular RNA and Cell Surface GlycoRNAs. Annual review of genomics and human genetics. 24:85-107. Pubmed: 37068783 DOI:10.1146/annurev-genom-101722-101224 The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs. -
Spencley AL, Bar S, Swigut T, Flynn RA, Lee CH, Chen LF, Bassik MC, Wysocka J. 2023. Co-transcriptional genome surveillance by HUSH is coupled to termination machinery. Molecular cell. 83(10):1623-1639.e8. Pubmed: 37164018 DOI:S1097-2765(23)00285-X Spencley AL, Bar S, Swigut T, Flynn RA, Lee CH, Chen LF, Bassik MC, Wysocka J. 2023. Co-transcriptional genome surveillance by HUSH is coupled to termination machinery. Molecular cell. 83(10):1623-1639.e8. Pubmed: 37164018 DOI:S1097-2765(23)00285-X The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Aryan F, Detrés D, Luo CC, Kim SX, Shah AN, Bartusel M, Flynn RA, Calo E. 2023. Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing. Molecular cell. 83(23):4413-4423.e10. Pubmed: 37979585 DOI:S1097-2765(23)00868-7 Aryan F, Detrés D, Luo CC, Kim SX, Shah AN, Bartusel M, Flynn RA, Calo E. 2023. Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing. Molecular cell. 83(23):4413-4423.e10. Pubmed: 37979585 DOI:S1097-2765(23)00868-7 DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.Copyright © 2023 Elsevier Inc. All rights reserved. 2022
-
Richards CM, Jabs S, Qiao W, Varanese LD, Schweizer M, Mosen PR, Riley NM, Klüssendorf M, Zengel JR, Flynn RA, Rustagi A, Widen JC, Peters CE, Ooi YS, Xie X, Shi PY, Bartenschlager R, Puschnik AS, Bogyo M, Bertozzi CR, Blish CA, Winter D, Nagamine CM, Braulke T, Carette JE. 2022. The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.). 378(6615):eabn5648. Pubmed: 36074821 DOI:10.1126/science.abn5648 Richards CM, Jabs S, Qiao W, Varanese LD, Schweizer M, Mosen PR, Riley NM, Klüssendorf M, Zengel JR, Flynn RA, Rustagi A, Widen JC, Peters CE, Ooi YS, Xie X, Shi PY, Bartenschlager R, Puschnik AS, Bogyo M, Bertozzi CR, Blish CA, Winter D, Nagamine CM, Braulke T, Carette JE. 2022. The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.). 378(6615):eabn5648. Pubmed: 36074821 DOI:10.1126/science.abn5648 Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder. -
Thangavelu G, Zaiken MC, Mohamed FA, Flynn R, Du J, Rhee SY, Riddle MJ, Aguilar EG, Panoskaltsis-Mortari A, Sanders ME, Blazar BR. 2022. Targeting the Retinoid X Receptor Pathway Prevents and Ameliorates Murine Chronic Graft-Versus-Host Disease. Frontiers in immunology. 13:765319. Pubmed: 35359939 DOI:10.3389/fimmu.2022.765319 Thangavelu G, Zaiken MC, Mohamed FA, Flynn R, Du J, Rhee SY, Riddle MJ, Aguilar EG, Panoskaltsis-Mortari A, Sanders ME, Blazar BR. 2022. Targeting the Retinoid X Receptor Pathway Prevents and Ameliorates Murine Chronic Graft-Versus-Host Disease. Frontiers in immunology. 13:765319. Pubmed: 35359939 DOI:10.3389/fimmu.2022.765319 Most allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients receive peripheral blood stem cell grafts resulting in a 30%-70% incidence of chronic graft-versus-host disease (cGVHD), a major cause of mortality and morbidity in long-term survivors. While systemic steroids remain the standard of care for first-line therapy, patients may require long-term administration, and those with steroid-resistant or refractory cGVHD have a worse prognosis. Although durable and deep responses with second-line therapies can be achieved in some patients, there remains an urgent need for new therapies. In this study, we evaluated the efficacy of IRX4204, a novel agonist that activates RXRs and is in clinical trials for cancer treatment to prevent and treat cGVHD in two complementary murine models. In a major histocompatibility complex mismatched, non-sclerodermatous multiorgan system model with bronchiolitis obliterans, IRX4204 prevented and reversed cGVHD including associated pulmonary dysfunction with restoration of germinal center T-follicular helper: T-follicular regulatory cell balance. In a minor histocompatibility antigen disparate sclerodermatous model, IRX4204 treatment significantly prevented and ameliorated skin cGVHD by reducing Th1 and Th17 differentiation due to anti-inflammatory properties. Together, these results indicate that IRX4204 is a promising therapeutic option to treat cGVHD with bronchiolitis obliterans or sclerodermatous manifestations.Copyright © 2022 Thangavelu, Zaiken, Mohamed, Flynn, Du, Rhee, Riddle, Aguilar, Panoskaltsis-Mortari, Sanders and Blazar. -
England WE, Wang J, Chen S, Baldi P, Flynn RA, Spitale RC. 2022. An atlas of posttranslational modifications on RNA binding proteins. Nucleic acids research. 50(8):4329-4339. Pubmed: 35438783 DOI:10.1093/nar/gkac243 England WE, Wang J, Chen S, Baldi P, Flynn RA, Spitale RC. 2022. An atlas of posttranslational modifications on RNA binding proteins. Nucleic acids research. 50(8):4329-4339. Pubmed: 35438783 DOI:10.1093/nar/gkac243 RNA structure and function are intimately tied to RNA binding protein recognition and regulation. Posttranslational modifications are chemical modifications which can control protein biology. The role of PTMs in the regulation RBPs is not well understood, in part due to a lacking analysis of PTM deposition on RBPs. Herein, we present an analysis of posttranslational modifications (PTMs) on RNA binding proteins (RBPs; a PTM RBP Atlas). We curate published datasets and primary literature to understand the landscape of PTMs and use protein-protein interaction data to understand and potentially provide a framework for understanding which enzymes are controlling PTM deposition and removal on the RBP landscape. Intersection of our data with The Cancer Genome Atlas also provides researchers understanding of mutations that would alter PTM deposition. Additional characterization of the RNA-protein interface provided from in-cell UV crosslinking experiments provides a framework for hypotheses about which PTMs could be regulating RNA binding and thus RBP function. Finally, we provide an online database for our data that is easy to use for the community. It is our hope our efforts will provide researchers will an invaluable tool to test the function of PTMs controlling RBP function and thus RNA biology.© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. -
Shivaprasad S, Weng KF, Ooi YS, Belk J, Carette JE, Flynn R, Sarnow P. 2022. Loquacious modulates flaviviral RNA replication in mosquito cells. PLoS pathogens. 18(4):e1010163. Pubmed: 35482886 DOI:10.1371/journal.ppat.1010163 Shivaprasad S, Weng KF, Ooi YS, Belk J, Carette JE, Flynn R, Sarnow P. 2022. Loquacious modulates flaviviral RNA replication in mosquito cells. PLoS pathogens. 18(4):e1010163. Pubmed: 35482886 DOI:10.1371/journal.ppat.1010163 Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells. -
Snyder JE, St-Aubin J, Yaddanapudi S, Marshall S, Strand S, Kruger S, Flynn R, Hyer DE. 2022. Reducing MRI-guided radiotherapy planning and delivery times via efficient leaf sequencing and segment shape optimization algorithms. Physics in medicine and biology. 67(5). Pubmed: 35130528 DOI:10.1088/1361-6560/ac5299 Snyder JE, St-Aubin J, Yaddanapudi S, Marshall S, Strand S, Kruger S, Flynn R, Hyer DE. 2022. Reducing MRI-guided radiotherapy planning and delivery times via efficient leaf sequencing and segment shape optimization algorithms. Physics in medicine and biology. 67(5). Pubmed: 35130528 DOI:10.1088/1361-6560/ac5299 ArrayCreative Commons Attribution license. 2021
-
Pluvinage JV, Sun J, Claes C, Flynn RA, Haney MS, Iram T, Meng X, Lindemann R, Riley NM, Danhash E, Chadarevian JP, Tapp E, Gate D, Kondapavulur S, Cobos I, Chetty S, Pașca AM, Pașca SP, Berry-Kravis E, Bertozzi CR, Blurton-Jones M, Wyss-Coray T. 2021. The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C. Science translational medicine. 13(622):eabg2919. Pubmed: 34851695 DOI:10.1126/scitranslmed.abg2919 Pluvinage JV, Sun J, Claes C, Flynn RA, Haney MS, Iram T, Meng X, Lindemann R, Riley NM, Danhash E, Chadarevian JP, Tapp E, Gate D, Kondapavulur S, Cobos I, Chetty S, Pașca AM, Pașca SP, Berry-Kravis E, Bertozzi CR, Blurton-Jones M, Wyss-Coray T. 2021. The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C. Science translational medicine. 13(622):eabg2919. Pubmed: 34851695 DOI:10.1126/scitranslmed.abg2919 Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid–dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate–binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human mutant induced pluripotent stem cell–derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC. -
Chan D, Feng C, England WE, Wyman D, Flynn RA, Wang X, Shi Y, Mortazavi A, Spitale RC. 2021. Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing. Nucleic acids research. 49(20):11868-11882. Pubmed: 34634799 DOI:10.1093/nar/gkab885 Chan D, Feng C, England WE, Wyman D, Flynn RA, Wang X, Shi Y, Mortazavi A, Spitale RC. 2021. Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing. Nucleic acids research. 49(20):11868-11882. Pubmed: 34634799 DOI:10.1093/nar/gkab885 RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptome-wide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA.© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. -
Corley M, Flynn RA, Blue SM, Yee BA, Chang HY, Yeo GW. 2021. fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins. STAR protocols. 2(3):100762. Pubmed: 34485935 DOI:10.1016/j.xpro.2021.100762 Corley M, Flynn RA, Blue SM, Yee BA, Chang HY, Yeo GW. 2021. fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins. STAR protocols. 2(3):100762. Pubmed: 34485935 DOI:10.1016/j.xpro.2021.100762 Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020).© 2021. -
Levy RB, Mousa HM, Lightbourn CO, Shiuey EJ, Latoni D, Duffort S, Flynn R, Du J, Barreras H, Zaiken M, Paz K, Blazar BR, Perez VL. 2021. Analyses and Correlation of Pathologic and Ocular Cutaneous Changes in Murine Graft versus Host Disease. International journal of molecular sciences. 23(1). Pubmed: 35008621 DOI:10.3390/ijms23010184 Levy RB, Mousa HM, Lightbourn CO, Shiuey EJ, Latoni D, Duffort S, Flynn R, Du J, Barreras H, Zaiken M, Paz K, Blazar BR, Perez VL. 2021. Analyses and Correlation of Pathologic and Ocular Cutaneous Changes in Murine Graft versus Host Disease. International journal of molecular sciences. 23(1). Pubmed: 35008621 DOI:10.3390/ijms23010184 Graft versus host disease (GVHD) is initiated by donor allo-reactive T cells activated against recipient antigens. Chronic GVHD (cGVHD) is characterized by immune responses that may resemble autoimmune features present in the scleroderma and Sjogren's syndrome. Unfortunately, ocular involvement occurs in approximately 60-90% of patients with cGVHD following allo-hematopoietic stem cell transplants (aHSCT). Ocular GVHD (oGVHD) may affect vision due to ocular adnexa damage leading to dry eye and keratopathy. Several other compartments including the skin are major targets of GVHD effector pathways. Using mouse aHSCT models, the objective was to characterize cGVHD associated alterations in the eye and skin to assess for correlations between these two organs. The examination of multiple models of MHC-matched and MHC-mismatched aHSCT identified a correlation between ocular and cutaneous involvement accompanying cGVHD. Studies detected a "positive" correlation, i.e., when cGVHD-induced ocular alterations were observed, cutaneous compartment alterations were also observed. When no or minimal ocular signs were detected, no or minimal skin changes were observed. In total, these findings suggest underlying cGVHD-inducing pathological immune mechanisms may be shared between the eye and skin. Based on the present observations, we posit that when skin involvement is present in aHSCT patients with cGVHD, the evaluation of the ocular surface by an ophthalmologist could potentially be of value. -
Flynn RA, Pedram K, Malaker SA, Batista PJ, Smith BAH, Johnson AG, George BM, Majzoub K, Villalta PW, Carette JE, Bertozzi CR. 2021. Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell. 184(12):3109-3124.e22. Pubmed: 34004145 DOI:S0092-8674(21)00503-1 Flynn RA, Pedram K, Malaker SA, Batista PJ, Smith BAH, Johnson AG, George BM, Majzoub K, Villalta PW, Carette JE, Bertozzi CR. 2021. Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell. 184(12):3109-3124.e22. Pubmed: 34004145 DOI:S0092-8674(21)00503-1 Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.Copyright © 2021 Elsevier Inc. All rights reserved. -
Flynn RA, Belk JA, Qi Y, Yasumoto Y, Wei J, Alfajaro MM, Shi Q, Mumbach MR, Limaye A, DeWeirdt PC, Schmitz CO, Parker KR, Woo E, Chang HY, Horvath TL, Carette JE, Bertozzi CR, Wilen CB, Satpathy AT. 2021. Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions. Cell. 184(9):2394-2411.e16. Pubmed: 33743211 DOI:S0092-8674(21)00297-X Flynn RA, Belk JA, Qi Y, Yasumoto Y, Wei J, Alfajaro MM, Shi Q, Mumbach MR, Limaye A, DeWeirdt PC, Schmitz CO, Parker KR, Woo E, Chang HY, Horvath TL, Carette JE, Bertozzi CR, Wilen CB, Satpathy AT. 2021. Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions. Cell. 184(9):2394-2411.e16. Pubmed: 33743211 DOI:S0092-8674(21)00297-X SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. 2020
-
Santoriello C, Sporrij A, Yang S, Flynn RA, Henriques T, Dorjsuren B, Custo Greig E, McCall W, Stanhope ME, Fazio M, Superdock M, Lichtig A, Adatto I, Abraham BJ, Kalocsay M, Jurynec M, Zhou Y, Adelman K, Calo E, Zon LI. 2020. RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature cell biology. 22(4):372-379. Pubmed: 32231306 DOI:10.1038/s41556-020-0493-0 Santoriello C, Sporrij A, Yang S, Flynn RA, Henriques T, Dorjsuren B, Custo Greig E, McCall W, Stanhope ME, Fazio M, Superdock M, Lichtig A, Adatto I, Abraham BJ, Kalocsay M, Jurynec M, Zhou Y, Adelman K, Calo E, Zon LI. 2020. RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature cell biology. 22(4):372-379. Pubmed: 32231306 DOI:10.1038/s41556-020-0493-0 The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. -
Salahudeen AA, Choi SS, Rustagi A, Zhu J, van Unen V, de la O SM, Flynn RA, Margalef-Català M, Santos AJM, Ju J, Batish A, Usui T, Zheng GXY, Edwards CE, Wagar LE, Luca V, Anchang B, Nagendran M, Nguyen K, Hart DJ, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Harbury PB, Glenn JS, Garcia KC, Davis MM, Baric RS, Sabatti C, Amieva MR, Blish CA, Desai TJ, Kuo CJ. 2020. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature. 588(7839):670-675. Pubmed: 33238290 DOI:10.1038/s41586-020-3014-1 Salahudeen AA, Choi SS, Rustagi A, Zhu J, van Unen V, de la O SM, Flynn RA, Margalef-Català M, Santos AJM, Ju J, Batish A, Usui T, Zheng GXY, Edwards CE, Wagar LE, Luca V, Anchang B, Nagendran M, Nguyen K, Hart DJ, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Harbury PB, Glenn JS, Garcia KC, Davis MM, Baric RS, Sabatti C, Amieva MR, Blish CA, Desai TJ, Kuo CJ. 2020. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature. 588(7839):670-675. Pubmed: 33238290 DOI:10.1038/s41586-020-3014-1 The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5 basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5 cells in basal organoids revealed a distinct population of ITGA6ITGB4 mitotic cells, whose offspring further segregated into a TNFRSF12A subfraction that comprised about ten per cent of KRT5 basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia. -
Corley M, Flynn RA, Lee B, Blue SM, Chang HY, Yeo GW. 2020. Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces. Molecular cell. 80(5):903-914.e8. Pubmed: 33242392 DOI:S1097-2765(20)30792-9 Corley M, Flynn RA, Lee B, Blue SM, Chang HY, Yeo GW. 2020. Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces. Molecular cell. 80(5):903-914.e8. Pubmed: 33242392 DOI:S1097-2765(20)30792-9 Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.Copyright © 2020 Elsevier Inc. All rights reserved. -
Zaro BW, Noh JJ, Mascetti VL, Demeter J, George B, Zukowska M, Gulati GS, Sinha R, Flynn RA, Banuelos A, Zhang A, Wilkinson AC, Jackson P, Weissman IL. 2020. Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells. eLife. 9. Pubmed: 33236985 DOI:10.7554/eLife.62210 Zaro BW, Noh JJ, Mascetti VL, Demeter J, George B, Zukowska M, Gulati GS, Sinha R, Flynn RA, Banuelos A, Zhang A, Wilkinson AC, Jackson P, Weissman IL. 2020. Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells. eLife. 9. Pubmed: 33236985 DOI:10.7554/eLife.62210 The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.© 2020, Zaro et al. -
Johnson AG, Flynn RA, Lapointe CP, Ooi YS, Zhao ML, Richards CM, Qiao W, Yamada SB, Couthouis J, Gitler AD, Carette JE, Puglisi JD. 2020. A memory of eS25 loss drives resistance phenotypes. Nucleic acids research. 48(13):7279-7297. Pubmed: 32463448 DOI:10.1093/nar/gkaa444 Johnson AG, Flynn RA, Lapointe CP, Ooi YS, Zhao ML, Richards CM, Qiao W, Yamada SB, Couthouis J, Gitler AD, Carette JE, Puglisi JD. 2020. A memory of eS25 loss drives resistance phenotypes. Nucleic acids research. 48(13):7279-7297. Pubmed: 32463448 DOI:10.1093/nar/gkaa444 In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. -
Flynn RA, Belk JA, Qi Y, Yasumoto Y, Schmitz CO, Mumbach MR, Limaye A, Wei J, Alfajaro MM, Parker KR, Chang HY, Horvath TL, Carette JE, Bertozzi C, Wilen CB, Satpathy AT. 2020. Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology. Pubmed: 33052334 DOI:10.1101/2020.10.06.327445 Flynn RA, Belk JA, Qi Y, Yasumoto Y, Schmitz CO, Mumbach MR, Limaye A, Wei J, Alfajaro MM, Parker KR, Chang HY, Horvath TL, Carette JE, Bertozzi C, Wilen CB, Satpathy AT. 2020. Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology. Pubmed: 33052334 DOI:10.1101/2020.10.06.327445 Array -
Thangavelu G, Du J, Paz KG, Loschi M, Zaiken MC, Flynn R, Taylor PA, Kirchmeier AK, Panoskaltsis-Mortari A, Luznik L, MacDonald KP, Hill GR, Maillard I, Munn DH, Serody JS, Murphy WJ, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Dahlberg C, Miller AT, Blazar BR. 2020. Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease. Blood. 135(1):28-40. Pubmed: 31697815 DOI:10.1182/blood.2019000032 Thangavelu G, Du J, Paz KG, Loschi M, Zaiken MC, Flynn R, Taylor PA, Kirchmeier AK, Panoskaltsis-Mortari A, Luznik L, MacDonald KP, Hill GR, Maillard I, Munn DH, Serody JS, Murphy WJ, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Dahlberg C, Miller AT, Blazar BR. 2020. Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease. Blood. 135(1):28-40. Pubmed: 31697815 DOI:10.1182/blood.2019000032 T-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell-mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb-/-) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb-/- donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.© 2020 by The American Society of Hematology. -
Shao Z, Flynn RA, Crowe JL, Zhu Y, Liang J, Jiang W, Aryan F, Aoude P, Bertozzi CR, Estes VM, Lee BJ, Bhagat G, Zha S, Calo E. 2020. DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature. 579(7798):291-296. Pubmed: 32103174 DOI:10.1038/s41586-020-2041-2 Shao Z, Flynn RA, Crowe JL, Zhu Y, Liang J, Jiang W, Aryan F, Aoude P, Bertozzi CR, Estes VM, Lee BJ, Bhagat G, Zha S, Calo E. 2020. DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature. 579(7798):291-296. Pubmed: 32103174 DOI:10.1038/s41586-020-2041-2 The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster. -
Salahudeen AA, Choi SS, Rustagi A, Zhu J, de la O SM, Flynn RA, Margalef-Català M, Santos AJM, Ju J, Batish A, van Unen V, Usui T, Zheng GXY, Edwards CE, Wagar LE, Luca V, Anchang B, Nagendran M, Nguyen K, Hart DJ, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Harbury PB, Glenn JS, Garcia KC, Davis MM, Baric RS, Sabatti C, Amieva MR, Blish CA, Desai TJ, Kuo CJ. 2020. Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures. bioRxiv : the preprint server for biology. Pubmed: 32743583 DOI:10.1101/2020.07.27.212076 Salahudeen AA, Choi SS, Rustagi A, Zhu J, de la O SM, Flynn RA, Margalef-Català M, Santos AJM, Ju J, Batish A, van Unen V, Usui T, Zheng GXY, Edwards CE, Wagar LE, Luca V, Anchang B, Nagendran M, Nguyen K, Hart DJ, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Harbury PB, Glenn JS, Garcia KC, Davis MM, Baric RS, Sabatti C, Amieva MR, Blish CA, Desai TJ, Kuo CJ. 2020. Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures. bioRxiv : the preprint server for biology. Pubmed: 32743583 DOI:10.1101/2020.07.27.212076 The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid cells contained a distinct mitotic population whose proliferation segregated to a subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A subset of FACS-purified ITGA6 ITGB4 basal cells from human lung or derivative organoids. In vivo, TNFRSF12A cells comprised ~10% of KRT5 basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia. 2019
-
Paz K, Flynn R, Du J, Qi J, Luznik L, Maillard I, MacDonald KP, Hill GR, Serody JS, Murphy WJ, Sage PT, Sharpe AH, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Bradner JE, Melnick AM, Blazar BR. 2019. Small-molecule BCL6 inhibitor effectively treats mice with nonsclerodermatous chronic graft-versus-host disease. Blood. 133(1):94-99. Pubmed: 30279226 DOI:10.1182/blood-2018-03-839993 Paz K, Flynn R, Du J, Qi J, Luznik L, Maillard I, MacDonald KP, Hill GR, Serody JS, Murphy WJ, Sage PT, Sharpe AH, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Bradner JE, Melnick AM, Blazar BR. 2019. Small-molecule BCL6 inhibitor effectively treats mice with nonsclerodermatous chronic graft-versus-host disease. Blood. 133(1):94-99. Pubmed: 30279226 DOI:10.1182/blood-2018-03-839993 Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.© 2019 by The American Society of Hematology. -
Mumbach MR, Granja JM, Flynn RA, Roake CM, Satpathy AT, Rubin AJ, Qi Y, Jiang Z, Shams S, Louie BH, Guo JK, Gennert DG, Corces MR, Khavari PA, Atianand MK, Artandi SE, Fitzgerald KA, Greenleaf WJ, Chang HY. 2019. HiChIRP reveals RNA-associated chromosome conformation. Nature methods. 16(6):489-492. Pubmed: 31133759 DOI:10.1038/s41592-019-0407-x Mumbach MR, Granja JM, Flynn RA, Roake CM, Satpathy AT, Rubin AJ, Qi Y, Jiang Z, Shams S, Louie BH, Guo JK, Gennert DG, Corces MR, Khavari PA, Atianand MK, Artandi SE, Fitzgerald KA, Greenleaf WJ, Chang HY. 2019. HiChIRP reveals RNA-associated chromosome conformation. Nature methods. 16(6):489-492. Pubmed: 31133759 DOI:10.1038/s41592-019-0407-x Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS). -
Paz K, Flynn R, Du J, Tannheimer S, Johnson AJ, Dong S, Stark AK, Okkenhaug K, Panoskaltsis-Mortari A, Sage PT, Sharpe AH, Luznik L, Ritz J, Soiffer RJ, Cutler CS, Koreth J, Antin JH, Miklos DB, MacDonald KP, Hill GR, Maillard I, Serody JS, Murphy WJ, Munn DH, Feser C, Zaiken M, Vanhaesebroeck B, Turka LA, Byrd JC, Blazar BR. 2019. Targeting PI3Kδ function for amelioration of murine chronic graft-versus-host disease. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19(6):1820-1830. Pubmed: 30748099 DOI:10.1111/ajt.15305 Paz K, Flynn R, Du J, Tannheimer S, Johnson AJ, Dong S, Stark AK, Okkenhaug K, Panoskaltsis-Mortari A, Sage PT, Sharpe AH, Luznik L, Ritz J, Soiffer RJ, Cutler CS, Koreth J, Antin JH, Miklos DB, MacDonald KP, Hill GR, Maillard I, Serody JS, Murphy WJ, Munn DH, Feser C, Zaiken M, Vanhaesebroeck B, Turka LA, Byrd JC, Blazar BR. 2019. Targeting PI3Kδ function for amelioration of murine chronic graft-versus-host disease. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19(6):1820-1830. Pubmed: 30748099 DOI:10.1111/ajt.15305 Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons. -
Ang CE, Ma Q, Wapinski OL, Fan S, Flynn RA, Lee QY, Coe B, Onoguchi M, Olmos VH, Do BT, Dukes-Rimsky L, Xu J, Tanabe K, Wang L, Elling U, Penninger JM, Zhao Y, Qu K, Eichler EE, Srivastava A, Wernig M, Chang HY. 2019. The novel lncRNA is pro-neurogenic and mutated in human neurodevelopmental disorders. eLife. 8. Pubmed: 30628890 DOI:10.7554/eLife.41770 Ang CE, Ma Q, Wapinski OL, Fan S, Flynn RA, Lee QY, Coe B, Onoguchi M, Olmos VH, Do BT, Dukes-Rimsky L, Xu J, Tanabe K, Wang L, Elling U, Penninger JM, Zhao Y, Qu K, Eichler EE, Srivastava A, Wernig M, Chang HY. 2019. The novel lncRNA is pro-neurogenic and mutated in human neurodevelopmental disorders. eLife. 8. Pubmed: 30628890 DOI:10.7554/eLife.41770 Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically . We further show that is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.© 2019, Ang et al. -
Sinha RK, Flynn R, Zaiken M, Paz K, Gavin AL, Nemazee D, Fernández JA, Xu X, Griffin JH, Blazar BR. 2019. Activated protein C ameliorates chronic graft-versus-host disease by PAR1-dependent biased cell signaling on T cells. Blood. 134(9):776-781. Pubmed: 31243040 DOI:10.1182/blood.2019001259 Sinha RK, Flynn R, Zaiken M, Paz K, Gavin AL, Nemazee D, Fernández JA, Xu X, Griffin JH, Blazar BR. 2019. Activated protein C ameliorates chronic graft-versus-host disease by PAR1-dependent biased cell signaling on T cells. Blood. 134(9):776-781. Pubmed: 31243040 DOI:10.1182/blood.2019001259 Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C's (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.© 2019 by The American Society of Hematology. -
Esfahani MS, Lee LJ, Jeon YJ, Flynn RA, Stehr H, Hui AB, Ishisoko N, Kildebeck E, Newman AM, Bratman SV, Porteus MH, Chang HY, Alizadeh AA, Diehn M. 2019. Functional significance of U2AF1 S34F mutations in lung adenocarcinomas. Nature communications. 10(1):5712. Pubmed: 31836708 DOI:10.1038/s41467-019-13392-y Esfahani MS, Lee LJ, Jeon YJ, Flynn RA, Stehr H, Hui AB, Ishisoko N, Kildebeck E, Newman AM, Bratman SV, Porteus MH, Chang HY, Alizadeh AA, Diehn M. 2019. Functional significance of U2AF1 S34F mutations in lung adenocarcinomas. Nature communications. 10(1):5712. Pubmed: 31836708 DOI:10.1038/s41467-019-13392-y The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in cross-linking at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD. -
Ooi YS, Majzoub K, Flynn RA, Mata MA, Diep J, Li JK, van Buuren N, Rumachik N, Johnson AG, Puschnik AS, Marceau CD, Mlera L, Grabowski JM, Kirkegaard K, Bloom ME, Sarnow P, Bertozzi CR, Carette JE. 2019. An RNA-centric dissection of host complexes controlling flavivirus infection. Nature microbiology. 4(12):2369-2382. Pubmed: 31384002 DOI:10.1038/s41564-019-0518-2 Ooi YS, Majzoub K, Flynn RA, Mata MA, Diep J, Li JK, van Buuren N, Rumachik N, Johnson AG, Puschnik AS, Marceau CD, Mlera L, Grabowski JM, Kirkegaard K, Bloom ME, Sarnow P, Bertozzi CR, Carette JE. 2019. An RNA-centric dissection of host complexes controlling flavivirus infection. Nature microbiology. 4(12):2369-2382. Pubmed: 31384002 DOI:10.1038/s41564-019-0518-2 Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses. 2018
-
Radojcic V, Paz K, Chung J, Du J, Perkey ET, Flynn R, Ivcevic S, Zaiken M, Friedman A, Yan M, Pletneva MA, Sarantopoulos S, Siebel CW, Blazar BR, Maillard I. 2018. Notch signaling mediated by Delta-like ligands 1 and 4 controls the pathogenesis of chronic GVHD in mice. Blood. 132(20):2188-2200. Pubmed: 30181175 DOI:10.1182/blood-2018-03-841155 Radojcic V, Paz K, Chung J, Du J, Perkey ET, Flynn R, Ivcevic S, Zaiken M, Friedman A, Yan M, Pletneva MA, Sarantopoulos S, Siebel CW, Blazar BR, Maillard I. 2018. Notch signaling mediated by Delta-like ligands 1 and 4 controls the pathogenesis of chronic GVHD in mice. Blood. 132(20):2188-2200. Pubmed: 30181175 DOI:10.1182/blood-2018-03-841155 Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2→BALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3 regulatory T cells. In the B6→B10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of and/or in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.© 2018 by The American Society of Hematology. -
Calo E, Gu B, Bowen ME, Aryan F, Zalc A, Liang J, Flynn RA, Swigut T, Chang HY, Attardi LD, Wysocka J. 2018. Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders. Nature. 554(7690):112-117. Pubmed: 29364875 DOI:10.1038/nature25449 Calo E, Gu B, Bowen ME, Aryan F, Zalc A, Liang J, Flynn RA, Swigut T, Chang HY, Attardi LD, Wysocka J. 2018. Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders. Nature. 554(7690):112-117. Pubmed: 29364875 DOI:10.1038/nature25449 Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations. -
Du J, Flynn R, Paz K, Ren HG, Ogata Y, Zhang Q, Gafken PR, Storer BE, Roy NH, Burkhardt JK, Mathews W, Tolar J, Lee SJ, Blazar BR, Paczesny S. 2018. Murine chronic graft-versus-host disease proteome profiling discovers CCL15 as a novel biomarker in patients. Blood. 131(15):1743-1754. Pubmed: 29348127 DOI:10.1182/blood-2017-08-800623 Du J, Flynn R, Paz K, Ren HG, Ogata Y, Zhang Q, Gafken PR, Storer BE, Roy NH, Burkhardt JK, Mathews W, Tolar J, Lee SJ, Blazar BR, Paczesny S. 2018. Murine chronic graft-versus-host disease proteome profiling discovers CCL15 as a novel biomarker in patients. Blood. 131(15):1743-1754. Pubmed: 29348127 DOI:10.1182/blood-2017-08-800623 Improved diagnostic and treatment methods are needed for chronic graft-versus-host disease (cGVHD), the leading cause of late nonrelapse mortality (NRM) in long-term survivors of allogenic hematopoietic cell transplantation. Validated biomarkers that facilitate disease diagnosis and classification generally are lacking in cGVHD. Here, we conducted whole serum proteomics analysis of a well-established murine multiorgan system cGVHD model. We discovered 4 upregulated proteins during cGVHD that are targetable by genetic ablation or blocking antibodies, including the RAS and JUN kinase activator, CRKL, and CXCL7, CCL8, and CCL9 chemokines. Donor T cells lacking CRK/CRKL prevented the generation of cGVHD, germinal center reactions, and macrophage infiltration seen with wild-type T cells. Whereas antibody blockade of CCL8 or CXCL7 was ineffective in treating cGVHD, CCL9 blockade reversed cGVHD clinical manifestations, histopathological changes, and immunopathological hallmarks. Mechanistically, elevated CCL9 expression was present predominantly in vascular smooth muscle cells and uniquely seen in cGVHD mice. Plasma concentrations of CCL15, the human homolog of mouse CCL9, were elevated in a previously published cohort of 211 cGVHD patients compared with controls and associated with NRM. In a cohort of 792 patients, CCL15 measured at day +100 could not predict cGVHD occurring within the next 3 months with clinically relevant sensitivity/specificity. Our findings demonstrate for the first time the utility of preclinical proteomics screening to identify potential new targets for cGVHD and specifically CCL15 as a diagnosis marker for cGVHD. These data warrant prospective biomarker validation studies.© 2018 by The American Society of Hematology. -
Verghese DA, Chun N, Paz K, Fribourg M, Woodruff TM, Flynn R, Hu Y, Xiong H, Zhang W, Yi Z, Du J, Blazar BR, Heeger PS. 2018. C5aR1 regulates T follicular helper differentiation and chronic graft-versus-host disease bronchiolitis obliterans. JCI insight. 3(24). Pubmed: 30568034 DOI:10.1172/jci.insight.124646 Verghese DA, Chun N, Paz K, Fribourg M, Woodruff TM, Flynn R, Hu Y, Xiong H, Zhang W, Yi Z, Du J, Blazar BR, Heeger PS. 2018. C5aR1 regulates T follicular helper differentiation and chronic graft-versus-host disease bronchiolitis obliterans. JCI insight. 3(24). Pubmed: 30568034 DOI:10.1172/jci.insight.124646 CD4+ follicular helper T (Tfh) cells are specialized providers of T cell help to B cells and can function as pathogenic mediators of murine antibody-dependent chronic graft-versus-host disease (GvHD). Using a parent→F1 model of lupus-like chronic GvHD, in which Tfh cell and germinal center (GC) B cell differentiation occurs over 14 days, we demonstrate that absence of CD4+ T cell-expressed C5a receptor 1 (C5ar1) or pharmacological C5aR1 blockade abrogated generation/expansion of Tfh cells, GC B cells, and autoantibodies. In a Tfh cell-dependent model of chronic GvHD manifested by bronchiolitis obliterans syndrome (BOS), C5aR1 antagonism initiated in mice with established disease ameliorated BOS and abolished the associated differentiation of Tfh and GC B cells. Guided by RNA-sequencing data, mechanistic studies performed using murine and human T cells showed that C5aR1 signaling amplifies IL-6-dependent expression of the transcription factor c-MAF and the cytokine IL-21 via phosphorylating phosphokinase B (AKT) and activating the mammalian target of rapamycin (mTOR). In addition to linking C5aR1-initiated signaling to Tfh cell differentiation, our findings suggest that C5aR1 may be a useful therapeutic target for prevention and/or treatment of individuals with Tfh cell-dependent diseases, including those chronic GvHD patients who have anti-host reactive antibodies. -
Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2018. Retraction Note: DDX5 and its associated lncRNA Rmrp modulate T17 cell effector functions. Nature. 562(7725):150. Pubmed: 29973715 DOI:10.1038/s41586-018-0311-z Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2018. Retraction Note: DDX5 and its associated lncRNA Rmrp modulate T17 cell effector functions. Nature. 562(7725):150. Pubmed: 29973715 DOI:10.1038/s41586-018-0311-z Change History: This Article has been retracted; see accompanying Retraction. Corrected online 20 January: In this Article, author Frank Rigo was incorrectly listed with a middle initial; this has been corrected in the online versions of the paper. 2017
-
Lee B, Flynn RA, Kadina A, Guo JK, Kool ET, Chang HY. 2017. Comparison of SHAPE reagents for mapping RNA structures inside living cells. RNA (New York, N.Y.). 23(2):169-174. Pubmed: 27879433 DOI:10.1261/rna.058784.116 Lee B, Flynn RA, Kadina A, Guo JK, Kool ET, Chang HY. 2017. Comparison of SHAPE reagents for mapping RNA structures inside living cells. RNA (New York, N.Y.). 23(2):169-174. Pubmed: 27879433 DOI:10.1261/rna.058784.116 Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N and 1M7 reagents, respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.© 2017 Lee et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society. -
Tomlin FM, Gerling-Driessen UIM, Liu YC, Flynn RA, Vangala JR, Lentz CS, Clauder-Muenster S, Jakob P, Mueller WF, Ordoñez-Rueda D, Paulsen M, Matsui N, Foley D, Rafalko A, Suzuki T, Bogyo M, Steinmetz LM, Radhakrishnan SK, Bertozzi CR. 2017. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS central science. 3(11):1143-1155. Pubmed: 29202016 DOI:10.1021/acscentsci.7b00224 Tomlin FM, Gerling-Driessen UIM, Liu YC, Flynn RA, Vangala JR, Lentz CS, Clauder-Muenster S, Jakob P, Mueller WF, Ordoñez-Rueda D, Paulsen M, Matsui N, Foley D, Rafalko A, Suzuki T, Bogyo M, Steinmetz LM, Radhakrishnan SK, Bertozzi CR. 2017. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS central science. 3(11):1143-1155. Pubmed: 29202016 DOI:10.1021/acscentsci.7b00224 Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis. -
Du J, Paz K, Thangavelu G, Schneidawind D, Baker J, Flynn R, Duramad O, Feser C, Panoskaltsis-Mortari A, Negrin RS, Blazar BR. 2017. Invariant natural killer T cells ameliorate murine chronic GVHD by expanding donor regulatory T cells. Blood. 129(23):3121-3125. Pubmed: 28416503 DOI:10.1182/blood-2016-11-752444 Du J, Paz K, Thangavelu G, Schneidawind D, Baker J, Flynn R, Duramad O, Feser C, Panoskaltsis-Mortari A, Negrin RS, Blazar BR. 2017. Invariant natural killer T cells ameliorate murine chronic GVHD by expanding donor regulatory T cells. Blood. 129(23):3121-3125. Pubmed: 28416503 DOI:10.1182/blood-2016-11-752444 Chronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typically with autoimmune-like features, resulting in high mortality and morbidity caused by treatment limitations. Invariant natural killer T cells (iNKTs), a small population characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious amounts of diverse cytokines on activation that exert potent immune regulatory function. Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by α-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.© 2017 by The American Society of Hematology. -
Simsek D, Tiu GC, Flynn RA, Byeon GW, Leppek K, Xu AF, Chang HY, Barna M. 2017. The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell. 169(6):1051-1065.e18. Pubmed: 28575669 DOI:S0092-8674(17)30582-2 Simsek D, Tiu GC, Flynn RA, Byeon GW, Leppek K, Xu AF, Chang HY, Barna M. 2017. The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell. 169(6):1051-1065.e18. Pubmed: 28575669 DOI:S0092-8674(17)30582-2 During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.Copyright © 2017 Elsevier Inc. All rights reserved. -
Chan D, Feng C, Zhen Y, Flynn RA, Spitale RC. 2017. Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts. Biochemistry. 56(13):1811-1814. Pubmed: 28319368 DOI:10.1021/acs.biochem.7b00128 Chan D, Feng C, Zhen Y, Flynn RA, Spitale RC. 2017. Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts. Biochemistry. 56(13):1811-1814. Pubmed: 28319368 DOI:10.1021/acs.biochem.7b00128 RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2'-hydroxyl acylation can then be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2'-hydroxyl. Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment. -
Mumbach MR, Satpathy AT, Boyle EA, Dai C, Gowen BG, Cho SW, Nguyen ML, Rubin AJ, Granja JM, Kazane KR, Wei Y, Nguyen T, Greenside PG, Corces MR, Tycko J, Simeonov DR, Suliman N, Li R, Xu J, Flynn RA, Kundaje A, Khavari PA, Marson A, Corn JE, Quertermous T, Greenleaf WJ, Chang HY. 2017. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics. 49(11):1602-1612. Pubmed: 28945252 DOI:10.1038/ng.3963 Mumbach MR, Satpathy AT, Boyle EA, Dai C, Gowen BG, Cho SW, Nguyen ML, Rubin AJ, Granja JM, Kazane KR, Wei Y, Nguyen T, Greenside PG, Corces MR, Tycko J, Simeonov DR, Suliman N, Li R, Xu J, Flynn RA, Kundaje A, Khavari PA, Marson A, Corn JE, Quertermous T, Greenleaf WJ, Chang HY. 2017. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics. 49(11):1602-1612. Pubmed: 28945252 DOI:10.1038/ng.3963 The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. -
Forcade E, Paz K, Flynn R, Griesenauer B, Amet T, Li W, Liu L, Bakoyannis G, Jiang D, Chu HW, Lobera M, Yang J, Wilkes DS, Du J, Gartlan K, Hill GR, MacDonald KP, Espada EL, Blanco P, Serody JS, Koreth J, Cutler CS, Antin JH, Soiffer RJ, Ritz J, Paczesny S, Blazar BR. 2017. An activated Th17-prone T cell subset involved in chronic graft-versus-host disease sensitive to pharmacological inhibition. JCI insight. 2(12). Pubmed: 28614794 DOI:10.1172/jci.insight.92111 Forcade E, Paz K, Flynn R, Griesenauer B, Amet T, Li W, Liu L, Bakoyannis G, Jiang D, Chu HW, Lobera M, Yang J, Wilkes DS, Du J, Gartlan K, Hill GR, MacDonald KP, Espada EL, Blanco P, Serody JS, Koreth J, Cutler CS, Antin JH, Soiffer RJ, Ritz J, Paczesny S, Blazar BR. 2017. An activated Th17-prone T cell subset involved in chronic graft-versus-host disease sensitive to pharmacological inhibition. JCI insight. 2(12). Pubmed: 28614794 DOI:10.1172/jci.insight.92111 Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic stem cell transplantation requiring novel therapies. CD146 and CCR5 are expressed by activated T cells and associated with increased T cell migration capacity and Th17 polarization. We performed a multiparametric flow cytometry analysis in a cohort of 40 HSCT patients together with a cGvHD murine model to understand the role of CD146-expressing subsets. We observed an increased frequency of CD146+ CD4 T cells in the 20 patients with active cGvHD with enhanced RORγt expression. This Th17-prone subset was enriched for cells coexpressing CD146 and CCR5 that harbor mixed Th1/Th17 features and were more frequent in cGvHD patients. Utilizing a murine cGvHD model with bronchiolitis obliterans (BO), we observed that donor T cells from CD146-deficient mice versus those from WT mice caused significantly reduced pulmonary cGvHD. Reduced cGvHD was not the result of failed germinal center B cell or T follicular helper cell generation. Instead, CD146-deficient T cells had significantly lower pulmonary macrophage infiltration and T cell CCR5, IL-17, and IFN-γ coexpression, suggesting defective pulmonary end-organ effector mechanisms. We, thus, evaluated the effect of TMP778, a small-molecule RORγt activity inhibitor. TMP778 markedly alleviated cGvHD in murine models similarly to agents targeting the Th17 pathway, such as STAT3 inhibitor or IL-17-blocking antibody. Our data suggest CD146-expressing T cells as a cGvHD biomarker and suggest that targeting the Th17 pathway may represent a promising therapy for cGvHD. 2016
-
Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.Copyright © 2016 Elsevier Inc. All rights reserved. -
Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY. 2016. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature methods. 13(11):919-922. Pubmed: 27643841 DOI:10.1038/nmeth.3999 Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY. 2016. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature methods. 13(11):919-922. Pubmed: 27643841 DOI:10.1038/nmeth.3999 Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C. -
Schmitt AM, Garcia JT, Hung T, Flynn RA, Shen Y, Qu K, Payumo AY, Peres-da-Silva A, Broz DK, Baum R, Guo S, Chen JK, Attardi LD, Chang HY. 2016. An inducible long noncoding RNA amplifies DNA damage signaling. Nature genetics. 48(11):1370-1376. Pubmed: 27668660 DOI:10.1038/ng.3673 Schmitt AM, Garcia JT, Hung T, Flynn RA, Shen Y, Qu K, Payumo AY, Peres-da-Silva A, Broz DK, Baum R, Guo S, Chen JK, Attardi LD, Chang HY. 2016. An inducible long noncoding RNA amplifies DNA damage signaling. Nature genetics. 48(11):1370-1376. Pubmed: 27668660 DOI:10.1038/ng.3673 Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks. -
Xu J, Spitale RC, Guan L, Flynn RA, Torre EA, Li R, Raber I, Qu K, Kern D, Knaggs HE, Chang HY, Chang AL. 2016. Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing. PloS one. 11(11):e0165913. Pubmed: 27829007 DOI:10.1371/journal.pone.0165913 Xu J, Spitale RC, Guan L, Flynn RA, Torre EA, Li R, Raber I, Qu K, Kern D, Knaggs HE, Chang HY, Chang AL. 2016. Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing. PloS one. 11(11):e0165913. Pubmed: 27829007 DOI:10.1371/journal.pone.0165913 While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18-89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3'-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans. -
Oh S, Flynn RA, Floor SN, Purzner J, Martin L, Do BT, Schubert S, Vaka D, Morrissy S, Li Y, Kool M, Hovestadt V, Jones DT, Northcott PA, Risch T, Warnatz HJ, Yaspo ML, Adams CM, Leib RD, Breese M, Marra MA, Malkin D, Lichter P, Doudna JA, Pfister SM, Taylor MD, Chang HY, Cho YJ. 2016. Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress. Oncotarget. 7(19):28169-82. Pubmed: 27058758 DOI:10.18632/oncotarget.8612 Oh S, Flynn RA, Floor SN, Purzner J, Martin L, Do BT, Schubert S, Vaka D, Morrissy S, Li Y, Kool M, Hovestadt V, Jones DT, Northcott PA, Risch T, Warnatz HJ, Yaspo ML, Adams CM, Leib RD, Breese M, Marra MA, Malkin D, Lichter P, Doudna JA, Pfister SM, Taylor MD, Chang HY, Cho YJ. 2016. Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress. Oncotarget. 7(19):28169-82. Pubmed: 27058758 DOI:10.18632/oncotarget.8612 DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation. -
Weiss JM, Chen W, Nyuydzefe MS, Trzeciak A, Flynn R, Tonra JR, Marusic S, Blazar BR, Waksal SD, Zanin-Zhorov A. 2016. ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings. Science signaling. 9(437):ra73. Pubmed: 27436361 DOI:10.1126/scisignal.aad8953 Weiss JM, Chen W, Nyuydzefe MS, Trzeciak A, Flynn R, Tonra JR, Marusic S, Blazar BR, Waksal SD, Zanin-Zhorov A. 2016. ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings. Science signaling. 9(437):ra73. Pubmed: 27436361 DOI:10.1126/scisignal.aad8953 Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.Copyright © 2016, American Association for the Advancement of Science. -
Zarnegar BJ, Flynn RA, Shen Y, Do BT, Chang HY, Khavari PA. 2016. irCLIP platform for efficient characterization of protein-RNA interactions. Nature methods. 13(6):489-92. Pubmed: 27111506 DOI:10.1038/nmeth.3840 Zarnegar BJ, Flynn RA, Shen Y, Do BT, Chang HY, Khavari PA. 2016. irCLIP platform for efficient characterization of protein-RNA interactions. Nature methods. 13(6):489-92. Pubmed: 27111506 DOI:10.1038/nmeth.3840 The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols. -
Li W, Liu L, Gomez A, Zhang J, Ramadan A, Zhang Q, Choi SW, Zhang P, Greenson JK, Liu C, Jiang D, Virts E, Kelich SL, Chu HW, Flynn R, Blazar BR, Hanenberg H, Hanash S, Paczesny S. 2016. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease. JCI insight. 1(6). Pubmed: 27195312 DOI:10.1172/jci.insight.86660 Li W, Liu L, Gomez A, Zhang J, Ramadan A, Zhang Q, Choi SW, Zhang P, Greenson JK, Liu C, Jiang D, Virts E, Kelich SL, Chu HW, Flynn R, Blazar BR, Hanenberg H, Hanash S, Paczesny S. 2016. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease. JCI insight. 1(6). Pubmed: 27195312 DOI:10.1172/jci.insight.86660 Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4CD146CCR5 T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4CD146CCR5 T cell population toward CCL14. Mice that received CD146 shRNA-transduced human T cells did not lose weight, showed better survival, and had fewer CD4CD146CCR5 T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA- transduced human T cells. Furthermore, the frequency of CD4CD146CCR5 Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. -
Flynn RA, Zhang QC, Spitale RC, Lee B, Mumbach MR, Chang HY. 2016. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature protocols. 11(2):273-90. Pubmed: 26766114 DOI:10.1038/nprot.2016.011 Flynn RA, Zhang QC, Spitale RC, Lee B, Mumbach MR, Chang HY. 2016. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature protocols. 11(2):273-90. Pubmed: 26766114 DOI:10.1038/nprot.2016.011 icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models. -
Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2016. Corrigendum: DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature. 533(7601):130. Pubmed: 26789242 DOI:10.1038/nature16968 Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2016. Corrigendum: DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature. 533(7601):130. Pubmed: 26789242 DOI:10.1038/nature16968 -
Flynn RA, Do BT, Rubin AJ, Calo E, Lee B, Kuchelmeister H, Rale M, Chu C, Kool ET, Wysocka J, Khavari PA, Chang HY. 2016. 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology. 23(3):231-8. Pubmed: 26878240 DOI:10.1038/nsmb.3176 Flynn RA, Do BT, Rubin AJ, Calo E, Lee B, Kuchelmeister H, Rale M, Chu C, Kool ET, Wysocka J, Khavari PA, Chang HY. 2016. 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology. 23(3):231-8. Pubmed: 26878240 DOI:10.1038/nsmb.3176 RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells, and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex, recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers, enhancers and promoters. -
Flynn R, Paz K, Du J, Reichenbach DK, Taylor PA, Panoskaltsis-Mortari A, Vulic A, Luznik L, MacDonald KK, Hill GR, Nyuydzefe MS, Weiss JM, Chen W, Trzeciak A, Serody JS, Aguilar EG, Murphy WJ, Maillard I, Munn D, Koreth J, Cutler CS, Antin JH, Ritz J, Waksal SD, Zanin-Zhorov A, Blazar BR. 2016. Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism. Blood. 127(17):2144-54. Pubmed: 26983850 DOI:10.1182/blood-2015-10-678706 Flynn R, Paz K, Du J, Reichenbach DK, Taylor PA, Panoskaltsis-Mortari A, Vulic A, Luznik L, MacDonald KK, Hill GR, Nyuydzefe MS, Weiss JM, Chen W, Trzeciak A, Serody JS, Aguilar EG, Murphy WJ, Maillard I, Munn D, Koreth J, Cutler CS, Antin JH, Ritz J, Waksal SD, Zanin-Zhorov A, Blazar BR. 2016. Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism. Blood. 127(17):2144-54. Pubmed: 26983850 DOI:10.1182/blood-2015-10-678706 Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.© 2016 by The American Society of Hematology. -
Zanin-Zhorov A, Flynn R, Waksal SD, Blazar BR. 2016. Isoform-specific targeting of ROCK proteins in immune cells. Small GTPases. 7(3):173-7. Pubmed: 27254302 DOI:10.1080/21541248.2016.1181698 Zanin-Zhorov A, Flynn R, Waksal SD, Blazar BR. 2016. Isoform-specific targeting of ROCK proteins in immune cells. Small GTPases. 7(3):173-7. Pubmed: 27254302 DOI:10.1080/21541248.2016.1181698 Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. -
Lu Z, Zhang QC, Lee B, Flynn RA, Smith MA, Robinson JT, Davidovich C, Gooding AR, Goodrich KJ, Mattick JS, Mesirov JP, Cech TR, Chang HY. 2016. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 165(5):1267-1279. Pubmed: 27180905 DOI:S0092-8674(16)30422-6 Lu Z, Zhang QC, Lee B, Flynn RA, Smith MA, Robinson JT, Davidovich C, Gooding AR, Goodrich KJ, Mattick JS, Mesirov JP, Cech TR, Chang HY. 2016. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 165(5):1267-1279. Pubmed: 27180905 DOI:S0092-8674(16)30422-6 RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.Copyright © 2016 Elsevier Inc. All rights reserved. 2015
-
Hechinger AK, Smith BA, Flynn R, Hanke K, McDonald-Hyman C, Taylor PA, Pfeifer D, Hackanson B, Leonhardt F, Prinz G, Dierbach H, Schmitt-Graeff A, Kovarik J, Blazar BR, Zeiser R. 2015. Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD. Blood. 125(3):570-80. Pubmed: 25352130 DOI:10.1182/blood-2014-06-581793 Hechinger AK, Smith BA, Flynn R, Hanke K, McDonald-Hyman C, Taylor PA, Pfeifer D, Hackanson B, Leonhardt F, Prinz G, Dierbach H, Schmitt-Graeff A, Kovarik J, Blazar BR, Zeiser R. 2015. Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD. Blood. 125(3):570-80. Pubmed: 25352130 DOI:10.1182/blood-2014-06-581793 The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.© 2015 by The American Society of Hematology. -
Mazumdar C, Shen Y, Xavy S, Zhao F, Reinisch A, Li R, Corces MR, Flynn RA, Buenrostro JD, Chan SM, Thomas D, Koenig JL, Hong WJ, Chang HY, Majeti R. 2015. Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell stem cell. 17(6):675-688. Pubmed: 26607380 DOI:S1934-5909(15)00424-5 Mazumdar C, Shen Y, Xavy S, Zhao F, Reinisch A, Li R, Corces MR, Flynn RA, Buenrostro JD, Chan SM, Thomas D, Koenig JL, Hong WJ, Chang HY, Majeti R. 2015. Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell stem cell. 17(6):675-688. Pubmed: 26607380 DOI:S1934-5909(15)00424-5 Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.Copyright © 2015 Elsevier Inc. All rights reserved. -
Flynn RA, Martin L, Spitale RC, Do BT, Sagan SM, Zarnegar B, Qu K, Khavari PA, Quake SR, Sarnow P, Chang HY. 2015. Dissecting noncoding and pathogen RNA-protein interactomes. RNA (New York, N.Y.). 21(1):135-43. Pubmed: 25411354 DOI:10.1261/rna.047803.114 Flynn RA, Martin L, Spitale RC, Do BT, Sagan SM, Zarnegar B, Qu K, Khavari PA, Quake SR, Sarnow P, Chang HY. 2015. Dissecting noncoding and pathogen RNA-protein interactomes. RNA (New York, N.Y.). 21(1):135-43. Pubmed: 25411354 DOI:10.1261/rna.047803.114 RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.© 2014 Flynn et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society. -
Blazar BR, Flynn R, Lee R, Marcucci G, Caliguiri MA, Heeger PS. 2015. Strategies to inhibit alloantibody production in alloprimed murine recipients of hematopoietic stem cell grafts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 15(4):931-41. Pubmed: 25762193 DOI:10.1111/ajt.13125 Blazar BR, Flynn R, Lee R, Marcucci G, Caliguiri MA, Heeger PS. 2015. Strategies to inhibit alloantibody production in alloprimed murine recipients of hematopoietic stem cell grafts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 15(4):931-41. Pubmed: 25762193 DOI:10.1111/ajt.13125 Alloantibody, not primed T cells, is the major barrier to bone marrow (BM) engraftment in allosensitized mice. We have shown that a single intravenous injection of donor splenocytes, to mimic a blood transfusion, results in high, sustained levels of serum alloantibody sufficient to eliminate donor BM within 3 h, resulting in uniform mortality in lethally irradiated allogeneic recipients. Current studies focused preventing and treating allopriming. Blockade of B cell survival signals with mTACI-Ig pre- and postpriming was ineffective, as was the B cell but not plasma cell depleting anti-CD20 mAb. Germinal center formation inhibition by lymphotoxin-beta receptor-Ig (LβR-Ig) diminished allosensitization, although conditional Prmd1 (Blimp-1) deletion in CD19+ cells was highly effective. By combining anti-CD20 mAb to reduce B cells and LTβR-Ig to diminish the frequency of B cells that could form germinal centers pre- and postpriming, allosensitization was precluded, permitting long-term survival in T- and NK-depleted, irradiated allogeneic recipients, whereas combined therapy postpriming alone was ineffective. As evidence of the critical role of B cells, the proteosomal inhibitor, bortezomib, given unencapsulated or encapsulated, proved ineffective in influencing allosensitization. These data extend our understanding of allopriming and provide a potential therapy for patients at risk for allosensitization and BM graft rejection.© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons. -
Calo E, Flynn RA, Martin L, Spitale RC, Chang HY, Wysocka J. 2015. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature. 518(7538):249-53. Pubmed: 25470060 DOI:10.1038/nature13923 Calo E, Flynn RA, Martin L, Spitale RC, Chang HY, Wysocka J. 2015. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature. 518(7538):249-53. Pubmed: 25470060 DOI:10.1038/nature13923 DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control. -
Chu C, Zhang QC, da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, Magnuson T, Heard E, Chang HY. 2015. Systematic discovery of Xist RNA binding proteins. Cell. 161(2):404-16. Pubmed: 25843628 DOI:S0092-8674(15)00312-8 Chu C, Zhang QC, da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, Magnuson T, Heard E, Chang HY. 2015. Systematic discovery of Xist RNA binding proteins. Cell. 161(2):404-16. Pubmed: 25843628 DOI:S0092-8674(15)00312-8 Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.Copyright © 2015 Elsevier Inc. All rights reserved. -
Veenstra RG, Flynn R, Kreymborg K, McDonald-Hyman C, Saha A, Taylor PA, Osborn MJ, Panoskaltsis-Mortari A, Schmitt-Graeff A, Lieberknecht E, Murphy WJ, Serody JS, Munn DH, Freeman GJ, Allison JP, Mak TW, van den Brink M, Zeiser R, Blazar BR. 2015. B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality. Blood. 125(21):3335-46. Pubmed: 25814530 DOI:10.1182/blood-2014-09-603357 Veenstra RG, Flynn R, Kreymborg K, McDonald-Hyman C, Saha A, Taylor PA, Osborn MJ, Panoskaltsis-Mortari A, Schmitt-Graeff A, Lieberknecht E, Murphy WJ, Serody JS, Munn DH, Freeman GJ, Allison JP, Mak TW, van den Brink M, Zeiser R, Blazar BR. 2015. B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality. Blood. 125(21):3335-46. Pubmed: 25814530 DOI:10.1182/blood-2014-09-603357 Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.© 2015 by The American Society of Hematology. -
Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY. 2015. Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 519(7544):486-90. Pubmed: 25799993 DOI:10.1038/nature14263 Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY. 2015. Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 519(7544):486-90. Pubmed: 25799993 DOI:10.1038/nature14263 Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression. -
Flynn R, Allen JL, Luznik L, MacDonald KP, Paz K, Alexander KA, Vulic A, Du J, Panoskaltsis-Mortari A, Taylor PA, Poe JC, Serody JS, Murphy WJ, Hill GR, Maillard I, Koreth J, Cutler CS, Soiffer RJ, Antin JH, Ritz J, Chao NJ, Clynes RA, Sarantopoulos S, Blazar BR. 2015. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease. Blood. 125(26):4085-94. Pubmed: 25852057 DOI:10.1182/blood-2014-08-595470 Flynn R, Allen JL, Luznik L, MacDonald KP, Paz K, Alexander KA, Vulic A, Du J, Panoskaltsis-Mortari A, Taylor PA, Poe JC, Serody JS, Murphy WJ, Hill GR, Maillard I, Koreth J, Cutler CS, Soiffer RJ, Antin JH, Ritz J, Chao NJ, Clynes RA, Sarantopoulos S, Blazar BR. 2015. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease. Blood. 125(26):4085-94. Pubmed: 25852057 DOI:10.1182/blood-2014-08-595470 Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.© 2015 by The American Society of Hematology. -
Zeiser R, Burchert A, Lengerke C, Verbeek M, Maas-Bauer K, Metzelder SK, Spoerl S, Ditschkowski M, Ecsedi M, Sockel K, Ayuk F, Ajib S, de Fontbrune FS, Na IK, Penter L, Holtick U, Wolf D, Schuler E, Meyer E, Apostolova P, Bertz H, Marks R, Lübbert M, Wäsch R, Scheid C, Stölzel F, Ordemann R, Bug G, Kobbe G, Negrin R, Brune M, Spyridonidis A, Schmitt-Gräff A, van der Velden W, Huls G, Mielke S, Grigoleit GU, Kuball J, Flynn R, Ihorst G, Du J, Blazar BR, Arnold R, Kröger N, Passweg J, Halter J, Socié G, Beelen D, Peschel C, Neubauer A, Finke J, Duyster J, von Bubnoff N. 2015. Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey. Leukemia. 29(10):2062-8. Pubmed: 26228813 DOI:10.1038/leu.2015.212 Zeiser R, Burchert A, Lengerke C, Verbeek M, Maas-Bauer K, Metzelder SK, Spoerl S, Ditschkowski M, Ecsedi M, Sockel K, Ayuk F, Ajib S, de Fontbrune FS, Na IK, Penter L, Holtick U, Wolf D, Schuler E, Meyer E, Apostolova P, Bertz H, Marks R, Lübbert M, Wäsch R, Scheid C, Stölzel F, Ordemann R, Bug G, Kobbe G, Negrin R, Brune M, Spyridonidis A, Schmitt-Gräff A, van der Velden W, Huls G, Mielke S, Grigoleit GU, Kuball J, Flynn R, Ihorst G, Du J, Blazar BR, Arnold R, Kröger N, Passweg J, Halter J, Socié G, Beelen D, Peschel C, Neubauer A, Finke J, Duyster J, von Bubnoff N. 2015. Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey. Leukemia. 29(10):2062-8. Pubmed: 26228813 DOI:10.1038/leu.2015.212 Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGVHD (1-10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3-90.7%, 95% confidence interval (CI)) and 97.4% (92.3-100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial. -
Grow EJ, Flynn RA, Chavez SL, Bayless NL, Wossidlo M, Wesche DJ, Martin L, Ware CB, Blish CA, Chang HY, Pera RA, Wysocka J. 2015. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature. 522(7555):221-5. Pubmed: 25896322 DOI:10.1038/nature14308 Grow EJ, Flynn RA, Chavez SL, Bayless NL, Wossidlo M, Wesche DJ, Martin L, Ware CB, Blish CA, Chang HY, Pera RA, Wysocka J. 2015. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature. 522(7555):221-5. Pubmed: 25896322 DOI:10.1038/nature14308 Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. -
Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2015. DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature. 528(7583):517-22. Pubmed: 26675721 DOI:10.1038/nature16193 Huang W, Thomas B, Flynn RA, Gavzy SJ, Wu L, Kim SV, Hall JA, Miraldi ER, Ng CP, Rigo F, Meadows S, Montoya NR, Herrera NG, Domingos AI, Rastinejad F, Myers RM, Fuller-Pace FV, Bonneau R, Chang HY, Acuto O, Littman DR. 2015. DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature. 528(7583):517-22. Pubmed: 26675721 DOI:10.1038/nature16193 T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases. -
Furlan SN, Watkins B, Tkachev V, Flynn R, Cooley S, Ramakrishnan S, Singh K, Giver C, Hamby K, Stempora L, Garrett A, Chen J, Betz KM, Ziegler CG, Tharp GK, Bosinger SE, Promislow DE, Miller JS, Waller EK, Blazar BR, Kean LS. 2015. Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention. Science translational medicine. 7(315):315ra191. Pubmed: 26606970 DOI:10.1126/scitranslmed.aad3231 Furlan SN, Watkins B, Tkachev V, Flynn R, Cooley S, Ramakrishnan S, Singh K, Giver C, Hamby K, Stempora L, Garrett A, Chen J, Betz KM, Ziegler CG, Tharp GK, Bosinger SE, Promislow DE, Miller JS, Waller EK, Blazar BR, Kean LS. 2015. Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention. Science translational medicine. 7(315):315ra191. Pubmed: 26606970 DOI:10.1126/scitranslmed.aad3231 Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3(+) T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.Copyright © 2015, American Association for the Advancement of Science. -
Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY. 2015. Erratum: Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 527(7577):264. Pubmed: 26416736 DOI:10.1038/nature15717 Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY. 2015. Erratum: Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 527(7577):264. Pubmed: 26416736 DOI:10.1038/nature15717 2014
-
Wan Y, Qu K, Zhang QC, Flynn RA, Manor O, Ouyang Z, Zhang J, Spitale RC, Snyder MP, Segal E, Chang HY. 2014. Landscape and variation of RNA secondary structure across the human transcriptome. Nature. 505(7485):706-9. Pubmed: 24476892 DOI:10.1038/nature12946 Wan Y, Qu K, Zhang QC, Flynn RA, Manor O, Ouyang Z, Zhang J, Spitale RC, Snyder MP, Segal E, Chang HY. 2014. Landscape and variation of RNA secondary structure across the human transcriptome. Nature. 505(7485):706-9. Pubmed: 24476892 DOI:10.1038/nature12946 In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation. -
Batista PJ, Molinie B, Wang J, Qu K, Zhang J, Li L, Bouley DM, Lujan E, Haddad B, Daneshvar K, Carter AC, Flynn RA, Zhou C, Lim KS, Dedon P, Wernig M, Mullen AC, Xing Y, Giallourakis CC, Chang HY. 2014. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell stem cell. 15(6):707-19. Pubmed: 25456834 DOI:S1934-5909(14)00451-2 Batista PJ, Molinie B, Wang J, Qu K, Zhang J, Li L, Bouley DM, Lujan E, Haddad B, Daneshvar K, Carter AC, Flynn RA, Zhou C, Lim KS, Dedon P, Wernig M, Mullen AC, Xing Y, Giallourakis CC, Chang HY. 2014. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell stem cell. 15(6):707-19. Pubmed: 25456834 DOI:S1934-5909(14)00451-2 N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.Copyright © 2014 Elsevier Inc. All rights reserved. -
Spitale RC, Flynn RA, Torre EA, Kool ET, Chang HY. 2014. RNA structural analysis by evolving SHAPE chemistry. Wiley interdisciplinary reviews. RNA. 5(6):867-81. Pubmed: 25132067 DOI:10.1002/wrna.1253 Spitale RC, Flynn RA, Torre EA, Kool ET, Chang HY. 2014. RNA structural analysis by evolving SHAPE chemistry. Wiley interdisciplinary reviews. RNA. 5(6):867-81. Pubmed: 25132067 DOI:10.1002/wrna.1253 RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.© 2014 John Wiley & Sons, Ltd. -
Alexander KA, Flynn R, Lineburg KE, Kuns RD, Teal BE, Olver SD, Lor M, Raffelt NC, Koyama M, Leveque L, Le Texier L, Melino M, Markey KA, Varelias A, Engwerda C, Serody JS, Janela B, Ginhoux F, Clouston AD, Blazar BR, Hill GR, MacDonald KP. 2014. CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease. The Journal of clinical investigation. 124(10):4266-80. Pubmed: 25157821 DOI:75935 Alexander KA, Flynn R, Lineburg KE, Kuns RD, Teal BE, Olver SD, Lor M, Raffelt NC, Koyama M, Leveque L, Le Texier L, Melino M, Markey KA, Varelias A, Engwerda C, Serody JS, Janela B, Ginhoux F, Clouston AD, Blazar BR, Hill GR, MacDonald KP. 2014. CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease. The Journal of clinical investigation. 124(10):4266-80. Pubmed: 25157821 DOI:75935 Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD. -
Dubovsky JA, Flynn R, Du J, Harrington BK, Zhong Y, Kaffenberger B, Yang C, Towns WH, Lehman A, Johnson AJ, Muthusamy N, Devine SM, Jaglowski S, Serody JS, Murphy WJ, Munn DH, Luznik L, Hill GR, Wong HK, MacDonald KK, Maillard I, Koreth J, Elias L, Cutler C, Soiffer RJ, Antin JH, Ritz J, Panoskaltsis-Mortari A, Byrd JC, Blazar BR. 2014. Ibrutinib treatment ameliorates murine chronic graft-versus-host disease. The Journal of clinical investigation. 124(11):4867-76. Pubmed: 25271622 DOI:75328 Dubovsky JA, Flynn R, Du J, Harrington BK, Zhong Y, Kaffenberger B, Yang C, Towns WH, Lehman A, Johnson AJ, Muthusamy N, Devine SM, Jaglowski S, Serody JS, Murphy WJ, Munn DH, Luznik L, Hill GR, Wong HK, MacDonald KK, Maillard I, Koreth J, Elias L, Cutler C, Soiffer RJ, Antin JH, Ritz J, Panoskaltsis-Mortari A, Byrd JC, Blazar BR. 2014. Ibrutinib treatment ameliorates murine chronic graft-versus-host disease. The Journal of clinical investigation. 124(11):4867-76. Pubmed: 25271622 DOI:75328 Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials. -
Paik KJ, Qu K, Hsueh B, Torre EA, Flynn RA, Chung MT, Spencley A, Wang KC, Wu JC, Longaker MT, Chang HY, Wan DC. 2014. Abstract 158: Identification of BMP-Responsive Long Noncoding RNAs in Pluripotent Cells. Plastic and reconstructive surgery. 133(3 Suppl):174. Pubmed: 25942268 DOI:10.1097/01.prs.0000444987.90678.b2 Paik KJ, Qu K, Hsueh B, Torre EA, Flynn RA, Chung MT, Spencley A, Wang KC, Wu JC, Longaker MT, Chang HY, Wan DC. 2014. Abstract 158: Identification of BMP-Responsive Long Noncoding RNAs in Pluripotent Cells. Plastic and reconstructive surgery. 133(3 Suppl):174. Pubmed: 25942268 DOI:10.1097/01.prs.0000444987.90678.b2 -
Yang YW, Flynn RA, Chen Y, Qu K, Wan B, Wang KC, Lei M, Chang HY. 2014. Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife. 3:e02046. Pubmed: 24521543 DOI:10.7554/eLife.02046 Yang YW, Flynn RA, Chen Y, Qu K, Wan B, Wang KC, Lei M, Chang HY. 2014. Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife. 3:e02046. Pubmed: 24521543 DOI:10.7554/eLife.02046 The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001. -
Flynn RA, Chang HY. 2014. Long noncoding RNAs in cell-fate programming and reprogramming. Cell stem cell. 14(6):752-61. Pubmed: 24905165 DOI:S1934-5909(14)00201-X Flynn RA, Chang HY. 2014. Long noncoding RNAs in cell-fate programming and reprogramming. Cell stem cell. 14(6):752-61. Pubmed: 24905165 DOI:S1934-5909(14)00201-X In recent years, long noncoding RNAs (lncRNAs) have emerged as an important class of regulators of gene expression. lncRNAs exhibit several distinctive features that confer unique regulatory functions, including exquisite cell- and tissue-specific expression and the capacity to transduce higher-order spatial information. Here we review evidence showing that lncRNAs exert critical functions in adult tissue stem cells, including skin, brain, and muscle, as well as in developmental patterning and pluripotency. We highlight new approaches for ascribing lncRNA functions and discuss mammalian dosage compensation as a classic example of an lncRNA network coupled to stem cell differentiation.Copyright © 2014 Elsevier Inc. All rights reserved. 2013
-
Spitale RC, Crisalli P, Flynn RA, Torre EA, Kool ET, Chang HY. 2013. RNA SHAPE analysis in living cells. Nature chemical biology. 9(1):18-20. Pubmed: 23178934 DOI:10.1038/nchembio.1131 Spitale RC, Crisalli P, Flynn RA, Torre EA, Kool ET, Chang HY. 2013. RNA SHAPE analysis in living cells. Nature chemical biology. 9(1):18-20. Pubmed: 23178934 DOI:10.1038/nchembio.1131 RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions. -
Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, Lee CS, Flockhart RJ, Groff AF, Chow J, Johnston D, Kim GE, Spitale RC, Flynn RA, Zheng GX, Aiyer S, Raj A, Rinn JL, Chang HY, Khavari PA. 2013. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature. 493(7431):231-5. Pubmed: 23201690 DOI:10.1038/nature11661 Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, Lee CS, Flockhart RJ, Groff AF, Chow J, Johnston D, Kim GE, Spitale RC, Flynn RA, Zheng GX, Aiyer S, Raj A, Rinn JL, Chang HY, Khavari PA. 2013. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature. 493(7431):231-5. Pubmed: 23201690 DOI:10.1038/nature11661 Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression. -
Hyun JS, Montoro DT, Lo DD, Flynn RA, Wong V, Chung MT, Longaker MT, Wan DC. 2013. The seed and the soil: optimizing stem cells and their environment for tissue regeneration. Annals of plastic surgery. 70(2):235-9. Pubmed: 23295233 DOI:10.1097/SAP.0b013e31826a18fb Hyun JS, Montoro DT, Lo DD, Flynn RA, Wong V, Chung MT, Longaker MT, Wan DC. 2013. The seed and the soil: optimizing stem cells and their environment for tissue regeneration. Annals of plastic surgery. 70(2):235-9. Pubmed: 23295233 DOI:10.1097/SAP.0b013e31826a18fb The potential for stem cells to serve as cellular building blocks for reconstruction of complex defects has prompted significant enthusiasm in the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly hinder their tissue forming capacity. To circumvent this obstacle, novel approaches have been developed to manipulate both the stem cell itself and its surrounding environmental niche. By understanding this paradigm of seed and soil optimization, innovative strategies may thus be developed to harness the true promise of stem cells for tissue regeneration. 2012
-
Yang W, Kim Y, Liu Y, Wu X, Flynn R. 2012. SU-E-T-547: Rotating Shield Brachytherapy (RSBT) for Cervical Cancer. Medical physics. 39(6Part18):3831. Pubmed: 28518488 DOI:10.1118/1.4735636 Yang W, Kim Y, Liu Y, Wu X, Flynn R. 2012. SU-E-T-547: Rotating Shield Brachytherapy (RSBT) for Cervical Cancer. Medical physics. 39(6Part18):3831. Pubmed: 28518488 DOI:10.1118/1.4735636 Array© 2012 American Association of Physicists in Medicine. -
Srinivasan M, Flynn R, Price A, Ranger A, Browning JL, Taylor PA, Ritz J, Antin JH, Murphy WJ, Luznik L, Shlomchik MJ, Panoskaltsis-Mortari A, Blazar BR. 2012. Donor B-cell alloantibody deposition and germinal center formation are required for the development of murine chronic GVHD and bronchiolitis obliterans. Blood. 119(6):1570-80. Pubmed: 22072556 DOI:10.1182/blood-2011-07-364414 Srinivasan M, Flynn R, Price A, Ranger A, Browning JL, Taylor PA, Ritz J, Antin JH, Murphy WJ, Luznik L, Shlomchik MJ, Panoskaltsis-Mortari A, Blazar BR. 2012. Donor B-cell alloantibody deposition and germinal center formation are required for the development of murine chronic GVHD and bronchiolitis obliterans. Blood. 119(6):1570-80. Pubmed: 22072556 DOI:10.1182/blood-2011-07-364414 Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4(+) T cells and B220(+) B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor-immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-β receptor-immunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy. -
Flynn RA, Chang HY. 2012. Active chromatin and noncoding RNAs: an intimate relationship. Current opinion in genetics & development. 22(2):172-8. Pubmed: 22154525 DOI:10.1016/j.gde.2011.11.002 Flynn RA, Chang HY. 2012. Active chromatin and noncoding RNAs: an intimate relationship. Current opinion in genetics & development. 22(2):172-8. Pubmed: 22154525 DOI:10.1016/j.gde.2011.11.002 Eukaryotic genomes are packaged into chromatin, where diverse histone modifications can demarcate chromatin domains that facilitate or block gene expression. While silent chromatin has been associated with long noncoding RNAs (lncRNAs) for some time, new studies suggest that noncoding RNAs also modulate the active chromatin state. Divergent, antisense, and enhancer-like intergenic noncoding RNAs can either activate or repress gene expression by altering histone H3 lysine 4 methylation. An emerging class of enhancer-like lncRNAs may link chromosome structure to chromatin state and establish active chromatin domains. The confluence of several new technologies promises to rapidly expand this fascinating topic of investigation.Copyright © 2011 Elsevier Ltd. All rights reserved. -
Liu Y, Flynn R, Yang W, Kim Y, Wu X. 2012. SU-E-T-606: Optimal Emission Angle Selection in Rotating Shield Brachytherapy. Medical physics. 39(6Part19):3845. Pubmed: 28517084 DOI:10.1118/1.4735695 Liu Y, Flynn R, Yang W, Kim Y, Wu X. 2012. SU-E-T-606: Optimal Emission Angle Selection in Rotating Shield Brachytherapy. Medical physics. 39(6Part19):3845. Pubmed: 28517084 DOI:10.1118/1.4735695 Array© 2012 American Association of Physicists in Medicine. -
Brunner AL, Beck AH, Edris B, Sweeney RT, Zhu SX, Li R, Montgomery K, Varma S, Gilks T, Guo X, Foley JW, Witten DM, Giacomini CP, Flynn RA, Pollack JR, Tibshirani R, Chang HY, van de Rijn M, West RB. 2012. Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers. Genome biology. 13(8):R75. Pubmed: 22929540 DOI:10.1186/gb-2012-13-8-r75 Brunner AL, Beck AH, Edris B, Sweeney RT, Zhu SX, Li R, Montgomery K, Varma S, Gilks T, Guo X, Foley JW, Witten DM, Giacomini CP, Flynn RA, Pollack JR, Tibshirani R, Chang HY, van de Rijn M, West RB. 2012. Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers. Genome biology. 13(8):R75. Pubmed: 22929540 DOI:10.1186/gb-2012-13-8-r75 Array -
Veenstra RG, Taylor PA, Zhou Q, Panoskaltsis-Mortari A, Hirashima M, Flynn R, Liu D, Anderson AC, Strom TB, Kuchroo VK, Blazar BR. 2012. Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells. Blood. 120(3):682-90. Pubmed: 22677125 DOI:10.1182/blood-2011-10-387977 Veenstra RG, Taylor PA, Zhou Q, Panoskaltsis-Mortari A, Hirashima M, Flynn R, Liu D, Anderson AC, Strom TB, Kuchroo VK, Blazar BR. 2012. Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells. Blood. 120(3):682-90. Pubmed: 22677125 DOI:10.1182/blood-2011-10-387977 T-cell immunoglobulin mucin-3 (Tim-3) is expressed on pathogenic T cells, and its ligand galectin-9 (gal-9) is up-regulated in inflamed tissues. When Tim-3(+) T cells encounter high gal-9 levels, they are deleted. Tim-3 is up-regulated on activated T cells during GVHD. Inhibition of Tim-3/gal-9 binding by infusion of a Tim-3-Ig fusion protein or Tim-3(-/-) donor T cells increased T-cell proliferation and GVHD lethality. When the Tim-3/gal-9 pathway engagement was augmented using gal-9 transgenic recipients, GVHD lethality was slowed. Together, these data indicate a potential for modulating this pathway to reduce disease by increasing Tim-3 or gal-9 engagement. Paradoxically, when Tim-3/gal-9 was inhibited in the absence of donor T-regulatory cells (Tregs), GVHD was inhibited. GVHD reduction was associated with decreased colonic inflammatory cytokines as well as epithelial barrier destruction. CD25-depleted Tim-3(-/-) donor T cells underwent increased activation-induced cell death because of increased IFN-γ production. To our knowledge, these studies are the first to show that although the absence of Tim-3/gal-9 pathway interactions augments systemic GVHD, concurrent donor Treg depletion paradoxically and surprisingly inhibits GVHD. Thus, although donor Tregs typically inhibit GVHD, under some conditions, such Tregs actually may contribute to GVHD by reducing activation-induced T-cell death. 2011
-
Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, Chen Y, Lajoie BR, Protacio A, Flynn RA, Gupta RA, Wysocka J, Lei M, Dekker J, Helms JA, Chang HY. 2011. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 472(7341):120-4. Pubmed: 21423168 DOI:10.1038/nature09819 Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, Chen Y, Lajoie BR, Protacio A, Flynn RA, Gupta RA, Wysocka J, Lei M, Dekker J, Helms JA, Chang HY. 2011. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 472(7341):120-4. Pubmed: 21423168 DOI:10.1038/nature09819 The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.©2011 Macmillan Publishers Limited. All rights reserved -
Dronadula N, Du L, Flynn R, Buckler J, Kho J, Jiang Z, Tanaka S, Dichek DA. 2011. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels. Gene therapy. 18(5):501-8. Pubmed: 21179172 DOI:10.1038/gt.2010.173 Dronadula N, Du L, Flynn R, Buckler J, Kho J, Jiang Z, Tanaka S, Dichek DA. 2011. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels. Gene therapy. 18(5):501-8. Pubmed: 21179172 DOI:10.1038/gt.2010.173 The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here, we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin (IL)-10) in large-vessel endothelial cells (ECs) at levels as high as obtained with the cytomegalovirus (CMV) immediate early promoter, while retaining EC specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely four copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining--and possibly enhancing--EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large-vessel EC, while minimizing systemic effects. -
Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J. 2011. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 470(7333):279-83. Pubmed: 21160473 DOI:10.1038/nature09692 Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J. 2011. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 470(7333):279-83. Pubmed: 21160473 DOI:10.1038/nature09692 Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis. -
Flynn RA, Almada AE, Zamudio JR, Sharp PA. 2011. Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome. Proceedings of the National Academy of Sciences of the United States of America. 108(26):10460-5. Pubmed: 21670248 DOI:10.1073/pnas.1106630108 Flynn RA, Almada AE, Zamudio JR, Sharp PA. 2011. Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome. Proceedings of the National Academy of Sciences of the United States of America. 108(26):10460-5. Pubmed: 21670248 DOI:10.1073/pnas.1106630108 Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5' termini and heterogeneous nonpolyadenylated 3' ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1-4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome. 2010
-
Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB, Sharp PA, Young RA. 2010. c-Myc regulates transcriptional pause release. Cell. 141(3):432-45. Pubmed: 20434984 DOI:10.1016/j.cell.2010.03.030 Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB, Sharp PA, Young RA. 2010. c-Myc regulates transcriptional pause release. Cell. 141(3):432-45. Pubmed: 20434984 DOI:10.1016/j.cell.2010.03.030 Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.2010 Elsevier Inc. All rights reserved. -
Huang PH, Miraldi ER, Xu AM, Kundukulam VA, Del Rosario AM, Flynn RA, Cavenee WK, Furnari FB, White FM. 2010. Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth. Molecular bioSystems. 6(7):1227-37. Pubmed: 20461251 DOI:10.1039/c001196g Huang PH, Miraldi ER, Xu AM, Kundukulam VA, Del Rosario AM, Flynn RA, Cavenee WK, Furnari FB, White FM. 2010. Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth. Molecular bioSystems. 6(7):1227-37. Pubmed: 20461251 DOI:10.1039/c001196g To evaluate the role of individual EGFR phosphorylation sites in activating components of the cellular signaling network we have performed a mass spectrometry-based analysis of the phosphotyrosine network downstream of site-specific EGFRvIII mutants, enabling quantification of network-level effects of site-specific point mutations. Mutation at Y845, Y1068 or Y1148 resulted in diminished receptor phosphorylation, while mutation at Y1173 led to increased phosphorylation on multiple EGFRvIII residues. Altered phosphorylation at the receptor was recapitulated in downstream signaling network activation levels, with Y1173F mutation leading to increased phosphorylation throughout the network. Computational modeling of GBM cell growth as a function of network phosphorylation levels highlights the Erk pathway as crucial for regulating EGFRvIII-driven U87MG GBM cell behavior, with the unexpected finding that Erk1/2 is negatively correlated to GBM cell growth. Genetic manipulation of this pathway supports the model, demonstrating that EGFRvIII-expressing U87MG GBM cells are sensitive to Erk activation levels. Additionally, we developed a model describing glioblastoma cell growth based on a reduced set of phosphoproteins, which represent potential candidates for future development as therapeutic targets for EGFRvIII-positive glioblastoma patients. 2008
-
Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, Flynn RA, Young RA, Sharp PA. 2008. Divergent transcription from active promoters. Science (New York, N.Y.). 322(5909):1849-51. Pubmed: 19056940 DOI:10.1126/science.1162253 Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, Flynn RA, Young RA, Sharp PA. 2008. Divergent transcription from active promoters. Science (New York, N.Y.). 322(5909):1849-51. Pubmed: 19056940 DOI:10.1126/science.1162253 Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site-associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation. 2007
-
Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK, Furnari FB, White FM. 2007. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proceedings of the National Academy of Sciences of the United States of America. 104(31):12867-72. Pubmed: 17646646 Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK, Furnari FB, White FM. 2007. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proceedings of the National Academy of Sciences of the United States of America. 104(31):12867-72. Pubmed: 17646646 Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) commonly found in GBMs that confers enhanced tumorigenic behavior. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have performed a large-scale analysis of EGFRvIII-activated phosphotyrosine-mediated signaling pathways and thereby have identified and quantified 99 phosphorylation sites on 69 proteins. Distinct signaling responses were observed as a function of titrated EGFRvIII receptor levels with the phosphatidylinositol 3-kinase pathway being dominant over the MAPK and STAT3 pathways at a high level of EGFRvIII expression. Within this data set, the activating phosphorylation site on the c-Met receptor was found to be highly responsive to EGFRvIII levels, indicating cross-activation of the c-Met receptor tyrosine kinase by EGFRvIII. To determine the significance of this finding, we devised a combined treatment regimen that used a c-Met kinase inhibitor and either an EGFR kinase inhibitor or cisplatin. This regimen resulted in enhanced cytotoxicity of EGFRvIII-expressing cells compared with treatment with either compound alone. These results suggest that the clinical use of c-Met kinase inhibitors in combination with either EGFR inhibitors or standard chemotherapeutics might represent a previously undescribed therapeutic approach to overcome the observed chemoresistance in patients with GBMs expressing EGFRvIII. 2006
-
Tuli R, Flynn RA, Brill KL, Sabol JL, Usuki KY, Rosenberg AL. 2006. Diagnosis, treatment, and management of breast cancer in previously augmented women. The breast journal. 12(4):343-8. Pubmed: 16848844 Tuli R, Flynn RA, Brill KL, Sabol JL, Usuki KY, Rosenberg AL. 2006. Diagnosis, treatment, and management of breast cancer in previously augmented women. The breast journal. 12(4):343-8. Pubmed: 16848844 Augmentation mammaplasty is rapidly becoming one of the most frequently performed cosmetic surgeries. However, as the augmented patient population ages, major concerns associated with the screening, diagnosis and treatment of breast cancer are being realized. Although current evidence convincingly indicates that breast implants do not play a role in inducing localized or systemic disease, particularly breast cancer, recent studies have shown implants not only reduce the sensitivity of mammography, but interfere with mammographic detection, possibly leading to delayed breast cancer diagnosis. In addition, the risk for local recurrence, as well as unfavorable cosmetic results, breast fibrosis, and capsular contracture following radiation therapy as part of breast-conserving therapy in previously augmented patients are of great concern. Given the overall lack of treatment consensus, paucity of literature, and increasing number of augmented breast cancer patients, we provide a retrospective review of the diagnosis, treatment, and follow-up of 12 augmented patients from 1998 to 2004 who developed breast cancer. Eight of 12 augmented patients presented with a palpable mass on physical examination, which prompted further mammographic evaluation. Abnormalities in the remaining four individuals were detected on routine mammographic screening. Pathology staging results were available for all 12 patients. Breast-conserving therapy was used to treat six patients and adequate negative pathologic margins were obtained in all patients. The remaining six patients were treated with mastectomy due to multifocal disease, inadequate margins, or proximity to the implant capsule. Thus far, one patient has had local recurrence and one patient has had distant recurrence after initial surgery. No evidence of local or systemic recurrence, infection, contracture, poor cosmetic outcome, or other complications has been detected in the remaining 10 patients as of the most recent follow-up. Based on this small cohort of augmented women, the presence of implants led to an increased proportion of palpable tumors, in spite of routine screening mammography. Consistent with other studies, although our results suggest a tendency toward delayed diagnosis in augmented women relative to age-matched controls, this did not appear to influence the overall prognosis. 1990
-
Kosslyn SM, Flynn RA, Amsterdam JB, Wang G. 1990. Components of high-level vision: a cognitive neuroscience analysis and accounts of neurological syndromes. Cognition. 34(3):203-77. Pubmed: 2183962 Kosslyn SM, Flynn RA, Amsterdam JB, Wang G. 1990. Components of high-level vision: a cognitive neuroscience analysis and accounts of neurological syndromes. Cognition. 34(3):203-77. Pubmed: 2183962 Neuroanatomical, neurophysiological, and computational constraints are used to motivate a set of hypotheses about the functional organization of high-level vision. A set of processing subsystems is posited that underlies the later stages of visual object recognition and identification; these subsystems have been implemented in a running computer simulation model. The model is damaged in a variety of ways, and its performance on a set of tasks is observed. Dysfunctions arise from disruptions of the subsystems, disruptions of their interconnections, compensations by intact subsystems, and diminished activational capacity. The most common dysfunctions of high-level vision following brain damage are then reviewed, and accounts are offered by reference to the stimulation model. According to the theory and model, each type of dysfunction can arise from numerous underlying causes, all of which are potentially distinguishable by empirical methods.