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Buenrostro Lab Publications
Featured Publications
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2019. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature biotechnology. 37(8):916-924. Pubmed: 31235917 DOI:10.1038/s41587-019-0147-6 Lareau CA, Duarte FM, Chew JG, Kartha VK, Burkett ZD, Kohlway AS, Pokholok D, Aryee MJ, Steemers FJ, Lebofsky R, Buenrostro JD. 2019. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature biotechnology. 37(8):916-924. Pubmed: 31235917 DOI:10.1038/s41587-019-0147-6 Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (10 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform. -
Shema E, Bernstein BE, Buenrostro JD. 2019. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nature genetics. 51(1):19-25. Pubmed: 30559489 DOI:10.1038/s41588-018-0290-x Shema E, Bernstein BE, Buenrostro JD. 2019. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nature genetics. 51(1):19-25. Pubmed: 30559489 DOI:10.1038/s41588-018-0290-x Recent advances in single-cell and single-molecule epigenomic technologies now enable the study of genome regulation and dynamics at unprecedented resolution. In this Perspective, we highlight some of these transformative technologies and discuss how they have been used to identify new modes of gene regulation. We also contrast these assays with recent advances in single-cell transcriptomics and argue for the essential role of epigenomic technologies in both understanding cellular diversity and discovering gene regulatory mechanisms. In addition, we provide our view on the next generation of biological tools that we expect will open new avenues for elucidating the fundamental principles of gene regulation. Overall, this Perspective motivates the use of these high-resolution epigenomic technologies for mapping cell states and understanding regulatory diversity at single-molecule resolution within single cells. -
Buenrostro JD, Corces MR, Lareau CA, Wu B, Schep AN, Aryee MJ, Majeti R, Chang HY, Greenleaf WJ. 2018. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell. 173(6):1535-1548.e16. Pubmed: 29706549 DOI:S0092-8674(18)30446-X Buenrostro JD, Corces MR, Lareau CA, Wu B, Schep AN, Aryee MJ, Majeti R, Chang HY, Greenleaf WJ. 2018. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell. 173(6):1535-1548.e16. Pubmed: 29706549 DOI:S0092-8674(18)30446-X Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.Copyright © 2018 Elsevier Inc. All rights reserved. -
Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, Snyder MP, Pritchard JK, Kundaje A, Greenleaf WJ, Majeti R, Chang HY. 2016. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics. 48(10):1193-203. Pubmed: 27526324 DOI:10.1038/ng.3646 Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, Snyder MP, Pritchard JK, Kundaje A, Greenleaf WJ, Majeti R, Chang HY. 2016. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics. 48(10):1193-203. Pubmed: 27526324 DOI:10.1038/ng.3646 We define the chromatin accessibility and transcriptional landscapes in 13 human primary blood cell types that span the hematopoietic hierarchy. Exploiting the finding that the enhancer landscape better reflects cell identity than mRNA levels, we enable 'enhancer cytometry' for enumeration of pure cell types from complex populations. We identify regulators governing hematopoietic differentiation and further show the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia (AML), chromatin accessibility uncovers unique regulatory evolution in cancer cells with a progressively increasing mutation burden. Single AML cells exhibit distinctive mixed regulome profiles corresponding to disparate developmental stages. A method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of preleukemic hematopoietic stem cell characteristics. Thus, regulome dynamics can provide diverse insights into hematopoietic development and disease. -
Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. 2015. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 523(7561):486-90. Pubmed: 26083756 DOI:10.1038/nature14590 Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. 2015. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 523(7561):486-90. Pubmed: 26083756 DOI:10.1038/nature14590 Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'. -
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods. 10(12):1213-8. Pubmed: 24097267 DOI:10.1038/nmeth.2688 Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods. 10(12):1213-8. Pubmed: 24097267 DOI:10.1038/nmeth.2688 We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.
All Publications
2023
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Otto JE, Ursu O, Wu AP, Winter EB, Cuoco MS, Ma S, Qian K, Michel BC, Buenrostro JD, Berger B, Regev A, Kadoch C. 2023. Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens. Molecular cell. 83(8):1350-1367.e7. Pubmed: 37028419 DOI:S1097-2765(23)00203-4 Otto JE, Ursu O, Wu AP, Winter EB, Cuoco MS, Ma S, Qian K, Michel BC, Buenrostro JD, Berger B, Regev A, Kadoch C. 2023. Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens. Molecular cell. 83(8):1350-1367.e7. Pubmed: 37028419 DOI:S1097-2765(23)00203-4 The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved. -
Joung J, Ma S, Tay T, Geiger-Schuller KR, Kirchgatterer PC, Verdine VK, Guo B, Arias-Garcia MA, Allen WE, Singh A, Kuksenko O, Abudayyeh OO, Gootenberg JS, Fu Z, Macrae RK, Buenrostro JD, Regev A, Zhang F. 2023. A transcription factor atlas of directed differentiation. Cell. 186(1):209-229.e26. Pubmed: 36608654 DOI:S0092-8674(22)01470-2 Joung J, Ma S, Tay T, Geiger-Schuller KR, Kirchgatterer PC, Verdine VK, Guo B, Arias-Garcia MA, Allen WE, Singh A, Kuksenko O, Abudayyeh OO, Gootenberg JS, Fu Z, Macrae RK, Buenrostro JD, Regev A, Zhang F. 2023. A transcription factor atlas of directed differentiation. Cell. 186(1):209-229.e26. Pubmed: 36608654 DOI:S0092-8674(22)01470-2 Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved. -
Hu Y, Ma S, Kartha VK, Duarte FM, Horlbeck M, Zhang R, Shrestha R, Labade A, Kletzien H, Meliki A, Castillo A, Durand N, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers A, Buenrostro JD. 2023. Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements. bioRxiv : the preprint server for biology. Pubmed: 37034577 DOI:10.1101/2023.03.28.533945 Hu Y, Ma S, Kartha VK, Duarte FM, Horlbeck M, Zhang R, Shrestha R, Labade A, Kletzien H, Meliki A, Castillo A, Durand N, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers A, Buenrostro JD. 2023. Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements. bioRxiv : the preprint server for biology. Pubmed: 37034577 DOI:10.1101/2023.03.28.533945 Array -
Morse DB, Michalowski AM, Ceribelli M, De Jonghe J, Vias M, Riley D, Davies-Hill T, Voss T, Pittaluga S, Muus C, Liu J, Boyle S, Weitz DA, Brenton JD, Buenrostro JD, Knowles TPJ, Thomas CJ. 2023. Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics. Cell systems. 14(6):464-481.e7. Pubmed: 37348462 DOI:S2405-4712(23)00147-3 Morse DB, Michalowski AM, Ceribelli M, De Jonghe J, Vias M, Riley D, Davies-Hill T, Voss T, Pittaluga S, Muus C, Liu J, Boyle S, Weitz DA, Brenton JD, Buenrostro JD, Knowles TPJ, Thomas CJ. 2023. Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics. Cell systems. 14(6):464-481.e7. Pubmed: 37348462 DOI:S2405-4712(23)00147-3 Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.Published by Elsevier Inc. -
Papathanasiou S, Mynhier NA, Liu S, Brunette G, Stokasimov E, Jacob E, Li L, Comenho C, van Steensel B, Buenrostro JD, Zhang CZ, Pellman D. 2023. Heritable transcriptional defects from aberrations of nuclear architecture. Nature. 619(7968):184-192. Pubmed: 37286600 DOI:10.1038/s41586-023-06157-7 Papathanasiou S, Mynhier NA, Liu S, Brunette G, Stokasimov E, Jacob E, Li L, Comenho C, van Steensel B, Buenrostro JD, Zhang CZ, Pellman D. 2023. Heritable transcriptional defects from aberrations of nuclear architecture. Nature. 619(7968):184-192. Pubmed: 37286600 DOI:10.1038/s41586-023-06157-7 Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.© 2023. The Author(s). -
Mangiameli SM, Chen H, Earl AS, Dobkin JA, Lesman D, Buenrostro JD, Chen F. 2023. Photoselective sequencing: microscopically guided genomic measurements with subcellular resolution. Nature methods. 20(5):686-694. Pubmed: 37106232 DOI:10.1038/s41592-023-01845-8 Mangiameli SM, Chen H, Earl AS, Dobkin JA, Lesman D, Buenrostro JD, Chen F. 2023. Photoselective sequencing: microscopically guided genomic measurements with subcellular resolution. Nature methods. 20(5):686-694. Pubmed: 37106232 DOI:10.1038/s41592-023-01845-8 In biological systems, spatial organization and function are interconnected. Here we present photoselective sequencing, a new method for genomic and epigenomic profiling within morphologically distinct regions. Starting with an intact biological specimen, photoselective sequencing uses targeted illumination to selectively unblock a photocaged fragment library, restricting the sequencing-based readout to microscopically identified spatial regions. We validate photoselective sequencing by measuring the chromatin accessibility profiles of fluorescently labeled cell types within the mouse brain and comparing with published data. Furthermore, by combining photoselective sequencing with a computational strategy for decomposing bulk accessibility profiles, we find that the oligodendrocyte-lineage-cell population is relatively enriched for oligodendrocyte-progenitor cells in the cortex versus the corpus callosum. Finally, we leverage photoselective sequencing at the subcellular scale to identify features of chromatin that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that photoselective sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc. 2022
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Yuan W, Ma S, Brown JR, Kim K, Murek V, Trastulla L, Meissner A, Lodato S, Shetty AS, Levin JZ, Buenrostro JD, Ziller MJ, Arlotta P. 2022. Temporally divergent regulatory mechanisms govern neuronal diversification and maturation in the mouse and marmoset neocortex. Nature neuroscience. 25(8):1049-1058. Pubmed: 35915179 DOI:10.1038/s41593-022-01123-4 Yuan W, Ma S, Brown JR, Kim K, Murek V, Trastulla L, Meissner A, Lodato S, Shetty AS, Levin JZ, Buenrostro JD, Ziller MJ, Arlotta P. 2022. Temporally divergent regulatory mechanisms govern neuronal diversification and maturation in the mouse and marmoset neocortex. Nature neuroscience. 25(8):1049-1058. Pubmed: 35915179 DOI:10.1038/s41593-022-01123-4 Mammalian neocortical neurons span one of the most diverse cell type spectra of any tissue. Cortical neurons are born during embryonic development, and their maturation extends into postnatal life. The regulatory strategies underlying progressive neuronal development and maturation remain unclear. Here we present an integrated single-cell epigenomic and transcriptional analysis of individual mouse and marmoset cortical neuron classes, spanning both early postmitotic stages of identity acquisition and later stages of neuronal plasticity and circuit integration. We found that, in both species, the regulatory strategies controlling early and late stages of pan-neuronal development diverge. Early postmitotic neurons use more widely shared and evolutionarily conserved molecular regulatory programs. In contrast, programs active during later neuronal maturation are more brain- and neuron-specific and more evolutionarily divergent. Our work uncovers a temporal shift in regulatory choices during neuronal diversification and maturation in both mice and marmosets, which likely reflects unique evolutionary constraints on distinct events of neuronal development in the neocortex.© 2022. The Author(s). -
Uzquiano A, Kedaigle AJ, Pigoni M, Paulsen B, Adiconis X, Kim K, Faits T, Nagaraja S, Antón-Bolaños N, Gerhardinger C, Tucewicz A, Murray E, Jin X, Buenrostro J, Chen F, Velasco S, Regev A, Levin JZ, Arlotta P. 2022. Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex. Cell. 185(20):3770-3788.e27. Pubmed: 36179669 DOI:S0092-8674(22)01168-0 Uzquiano A, Kedaigle AJ, Pigoni M, Paulsen B, Adiconis X, Kim K, Faits T, Nagaraja S, Antón-Bolaños N, Gerhardinger C, Tucewicz A, Murray E, Jin X, Buenrostro J, Chen F, Velasco S, Regev A, Levin JZ, Arlotta P. 2022. Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex. Cell. 185(20):3770-3788.e27. Pubmed: 36179669 DOI:S0092-8674(22)01168-0 Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.Copyright © 2022 Elsevier Inc. All rights reserved. -
Kartha VK, Duarte FM, Hu Y, Ma S, Chew JG, Lareau CA, Earl A, Burkett ZD, Kohlway AS, Lebofsky R, Buenrostro JD. 2022. Functional inference of gene regulation using single-cell multi-omics. Cell genomics. 2(9). Pubmed: 36204155 DOI:10.1016/j.xgen.2022.100166 Kartha VK, Duarte FM, Hu Y, Ma S, Chew JG, Lareau CA, Earl A, Burkett ZD, Kohlway AS, Lebofsky R, Buenrostro JD. 2022. Functional inference of gene regulation using single-cell multi-omics. Cell genomics. 2(9). Pubmed: 36204155 DOI:10.1016/j.xgen.2022.100166 Cells require coordinated control over gene expression when responding to environmental stimuli. Here we apply scATAC-seq and single-cell RNA sequencing (scRNA-seq) in resting and stimulated human blood cells. Collectively, we generate ~91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of the immunological response across cell types, stimuli, and time. Advancing tools to integrate multi-omics data, we develop functional inference of gene regulation (FigR), a framework to computationally pair scA-TAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene-regulatory networks (GRNs) to identify candidate transcription factor (TF) regulators. Utilizing these paired multi-omics data, we define domains of regulatory chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility and gene expression at timescales of minutes. Construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues. -
Zhao T, Chiang ZD, Morriss JW, LaFave LM, Murray EM, Del Priore I, Meli K, Lareau CA, Nadaf NM, Li J, Earl AS, Macosko EZ, Jacks T, Buenrostro JD, Chen F. 2022. Spatial genomics enables multi-modal study of clonal heterogeneity in tissues. Nature. 601(7891):85-91. Pubmed: 34912115 DOI:10.1038/s41586-021-04217-4 Zhao T, Chiang ZD, Morriss JW, LaFave LM, Murray EM, Del Priore I, Meli K, Lareau CA, Nadaf NM, Li J, Earl AS, Macosko EZ, Jacks T, Buenrostro JD, Chen F. 2022. Spatial genomics enables multi-modal study of clonal heterogeneity in tissues. Nature. 601(7891):85-91. Pubmed: 34912115 DOI:10.1038/s41586-021-04217-4 The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations as well as the makeup of the tumour microenvironment. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.© 2021. The Author(s), under exclusive licence to Springer Nature Limited. 2021
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Lareau CA, Ludwig LS, Muus C, Gohil SH, Zhao T, Chiang Z, Pelka K, Verboon JM, Luo W, Christian E, Rosebrock D, Getz G, Boland GM, Chen F, Buenrostro JD, Hacohen N, Wu CJ, Aryee MJ, Regev A, Sankaran VG. 2021. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nature biotechnology. 39(4):451-461. Pubmed: 32788668 DOI:10.1038/s41587-020-0645-6 Lareau CA, Ludwig LS, Muus C, Gohil SH, Zhao T, Chiang Z, Pelka K, Verboon JM, Luo W, Christian E, Rosebrock D, Getz G, Boland GM, Chen F, Buenrostro JD, Hacohen N, Wu CJ, Aryee MJ, Regev A, Sankaran VG. 2021. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nature biotechnology. 39(4):451-461. Pubmed: 32788668 DOI:10.1038/s41587-020-0645-6 Natural mitochondrial DNA (mtDNA) mutations enable the inference of clonal relationships among cells. mtDNA can be profiled along with measures of cell state, but has not yet been combined with the massively parallel approaches needed to tackle the complexity of human tissue. Here, we introduce a high-throughput, droplet-based mitochondrial single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), a method that combines high-confidence mtDNA mutation calling in thousands of single cells with their concomitant high-quality accessible chromatin profile. This enables the inference of mtDNA heteroplasmy, clonal relationships, cell state and accessible chromatin variation in individual cells. We reveal single-cell variation in heteroplasmy of a pathologic mtDNA variant, which we associate with intra-individual chromatin variability and clonal evolution. We clonally trace thousands of cells from cancers, linking epigenomic variability to subclonal evolution, and infer cellular dynamics of differentiating hematopoietic cells in vitro and in vivo. Taken together, our approach enables the study of cellular population dynamics and clonal properties in vivo. -
Payne AC, Chiang ZD, Reginato PL, Mangiameli SM, Murray EM, Yao CC, Markoulaki S, Earl AS, Labade AS, Jaenisch R, Church GM, Boyden ES, Buenrostro JD, Chen F. 2021. In situ genome sequencing resolves DNA sequence and structure in intact biological samples. Science (New York, N.Y.). 371(6532). Pubmed: 33384301 DOI:10.1126/science.aay3446 Payne AC, Chiang ZD, Reginato PL, Mangiameli SM, Murray EM, Yao CC, Markoulaki S, Earl AS, Labade AS, Jaenisch R, Church GM, Boyden ES, Buenrostro JD, Chen F. 2021. In situ genome sequencing resolves DNA sequence and structure in intact biological samples. Science (New York, N.Y.). 371(6532). Pubmed: 33384301 DOI:10.1126/science.aay3446 Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Li Q, Meissner TB, Wang F, Du Z, Ma S, Kshirsagar S, Tilburgs T, Buenrostro JD, Uesugi M, Strominger JL. 2021. ELF3 activated by a superenhancer and an autoregulatory feedback loop is required for high-level HLA-C expression on extravillous trophoblasts. Proceedings of the National Academy of Sciences of the United States of America. 118(9). Pubmed: 33622787 DOI:10.1073/pnas.2025512118 Li Q, Meissner TB, Wang F, Du Z, Ma S, Kshirsagar S, Tilburgs T, Buenrostro JD, Uesugi M, Strominger JL. 2021. ELF3 activated by a superenhancer and an autoregulatory feedback loop is required for high-level HLA-C expression on extravillous trophoblasts. Proceedings of the National Academy of Sciences of the United States of America. 118(9). Pubmed: 33622787 DOI:10.1073/pnas.2025512118 HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence.Copyright © 2021 the Author(s). Published by PNAS. -
Choi S, Zhang B, Ma S, Gonzalez-Celeiro M, Stein D, Jin X, Kim ST, Kang YL, Besnard A, Rezza A, Grisanti L, Buenrostro JD, Rendl M, Nahrendorf M, Sahay A, Hsu YC. 2021. Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence. Nature. 592(7854):428-432. Pubmed: 33790465 DOI:10.1038/s41586-021-03417-2 Choi S, Zhang B, Ma S, Gonzalez-Celeiro M, Stein D, Jin X, Kim ST, Kang YL, Besnard A, Rezza A, Grisanti L, Buenrostro JD, Rendl M, Nahrendorf M, Sahay A, Hsu YC. 2021. Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence. Nature. 592(7854):428-432. Pubmed: 33790465 DOI:10.1038/s41586-021-03417-2 Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here we report that the stress hormone corticosterone-which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans-regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term. -
Lal A, Chiang ZD, Yakovenko N, Duarte FM, Israeli J, Buenrostro JD. 2021. Deep learning-based enhancement of epigenomics data with AtacWorks. Nature communications. 12(1):1507. Pubmed: 33686069 DOI:10.1038/s41467-021-21765-5 Lal A, Chiang ZD, Yakovenko N, Duarte FM, Israeli J, Buenrostro JD. 2021. Deep learning-based enhancement of epigenomics data with AtacWorks. Nature communications. 12(1):1507. Pubmed: 33686069 DOI:10.1038/s41467-021-21765-5 ATAC-seq is a widely-applied assay used to measure genome-wide chromatin accessibility; however, its ability to detect active regulatory regions can depend on the depth of sequencing coverage and the signal-to-noise ratio. Here we introduce AtacWorks, a deep learning toolkit to denoise sequencing coverage and identify regulatory peaks at base-pair resolution from low cell count, low-coverage, or low-quality ATAC-seq data. Models trained by AtacWorks can detect peaks from cell types not seen in the training data, and are generalizable across diverse sample preparations and experimental platforms. We demonstrate that AtacWorks enhances the sensitivity of single-cell experiments by producing results on par with those of conventional methods using ~10 times as many cells, and further show that this framework can be adapted to enable cross-modality inference of protein-DNA interactions. Finally, we establish that AtacWorks can enable new biological discoveries by identifying active regulatory regions associated with lineage priming in rare subpopulations of hematopoietic stem cells. -
Rose SA, Wroblewska A, Dhainaut M, Yoshida H, Shaffer JM, Bektesevic A, Ben-Zvi B, Rhoads A, Kim EY, Yu B, Lavin Y, Merad M, Buenrostro JD, Brown BD. 2021. A microRNA expression and regulatory element activity atlas of the mouse immune system. Nature immunology. 22(7):914-927. Pubmed: 34099919 DOI:10.1038/s41590-021-00944-y Rose SA, Wroblewska A, Dhainaut M, Yoshida H, Shaffer JM, Bektesevic A, Ben-Zvi B, Rhoads A, Kim EY, Yu B, Lavin Y, Merad M, Buenrostro JD, Brown BD. 2021. A microRNA expression and regulatory element activity atlas of the mouse immune system. Nature immunology. 22(7):914-927. Pubmed: 34099919 DOI:10.1038/s41590-021-00944-y To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system. -
Avagyan S, Weber MC, Ma S, Prasad M, Mannherz WP, Yang S, Buenrostro JD, Zon LI. 2021. Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages. Blood advances. 5(13):2673-2686. Pubmed: 34170284 DOI:10.1182/bloodadvances.2020002992 Avagyan S, Weber MC, Ma S, Prasad M, Mannherz WP, Yang S, Buenrostro JD, Zon LI. 2021. Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages. Blood advances. 5(13):2673-2686. Pubmed: 34170284 DOI:10.1182/bloodadvances.2020002992 Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.© 2021 by The American Society of Hematology. -
Biancalani T, Scalia G, Buffoni L, Avasthi R, Lu Z, Sanger A, Tokcan N, Vanderburg CR, Segerstolpe Å, Zhang M, Avraham-Davidi I, Vickovic S, Nitzan M, Ma S, Subramanian A, Lipinski M, Buenrostro J, Brown NB, Fanelli D, Zhuang X, Macosko EZ, Regev A. 2021. Deep learning and alignment of spatially resolved single-cell transcriptomes with Tangram. Nature methods. 18(11):1352-1362. Pubmed: 34711971 DOI:10.1038/s41592-021-01264-7 Biancalani T, Scalia G, Buffoni L, Avasthi R, Lu Z, Sanger A, Tokcan N, Vanderburg CR, Segerstolpe Å, Zhang M, Avraham-Davidi I, Vickovic S, Nitzan M, Ma S, Subramanian A, Lipinski M, Buenrostro J, Brown NB, Fanelli D, Zhuang X, Macosko EZ, Regev A. 2021. Deep learning and alignment of spatially resolved single-cell transcriptomes with Tangram. Nature methods. 18(11):1352-1362. Pubmed: 34711971 DOI:10.1038/s41592-021-01264-7 Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.© 2021. The Author(s). -
LaFave LM, Buenrostro JD. 2021. Unlocking PDAC initiation with AP-1. Nature cancer. 2(1):14-15. Pubmed: 35121892 DOI:10.1038/s43018-020-00158-5 LaFave LM, Buenrostro JD. 2021. Unlocking PDAC initiation with AP-1. Nature cancer. 2(1):14-15. Pubmed: 35121892 DOI:10.1038/s43018-020-00158-5 -
Del Priore I, Ma S, Strecker J, Jacks T, LaFave LM, Buenrostro JD. 2021. Protocol for single-cell ATAC sequencing using combinatorial indexing in mouse lung adenocarcinoma. STAR protocols. 2(2):100583. Pubmed: 34142101 DOI:10.1016/j.xpro.2021.100583 Del Priore I, Ma S, Strecker J, Jacks T, LaFave LM, Buenrostro JD. 2021. Protocol for single-cell ATAC sequencing using combinatorial indexing in mouse lung adenocarcinoma. STAR protocols. 2(2):100583. Pubmed: 34142101 DOI:10.1016/j.xpro.2021.100583 Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples. For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020).© 2021 The Authors. 2020
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Ma S, Zhang B, LaFave LM, Earl AS, Chiang Z, Hu Y, Ding J, Brack A, Kartha VK, Tay T, Law T, Lareau C, Hsu YC, Regev A, Buenrostro JD. 2020. Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin. Cell. 183(4):1103-1116.e20. Pubmed: 33098772 DOI:S0092-8674(20)31253-8 Ma S, Zhang B, LaFave LM, Earl AS, Chiang Z, Hu Y, Ding J, Brack A, Kartha VK, Tay T, Law T, Lareau C, Hsu YC, Regev A, Buenrostro JD. 2020. Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin. Cell. 183(4):1103-1116.e20. Pubmed: 33098772 DOI:S0092-8674(20)31253-8 Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.Copyright © 2020 Elsevier Inc. All rights reserved. -
Agbleke AA, Amitai A, Buenrostro JD, Chakrabarti A, Chu L, Hansen AS, Koenig KM, Labade AS, Liu S, Nozaki T, Ovchinnikov S, Seeber A, Shaban HA, Spille JH, Stephens AD, Su JH, Wadduwage D. 2020. Advances in Chromatin and Chromosome Research: Perspectives from Multiple Fields. Molecular cell. 79(6):881-901. Pubmed: 32768408 DOI:S1097-2765(20)30469-X Agbleke AA, Amitai A, Buenrostro JD, Chakrabarti A, Chu L, Hansen AS, Koenig KM, Labade AS, Liu S, Nozaki T, Ovchinnikov S, Seeber A, Shaban HA, Spille JH, Stephens AD, Su JH, Wadduwage D. 2020. Advances in Chromatin and Chromosome Research: Perspectives from Multiple Fields. Molecular cell. 79(6):881-901. Pubmed: 32768408 DOI:S1097-2765(20)30469-X Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of chromatin underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects, which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology and to test concepts such as phase separation of chromatin.Copyright © 2020 Elsevier Inc. All rights reserved. -
LaFave LM, Kartha VK, Ma S, Meli K, Del Priore I, Lareau C, Naranjo S, Westcott PMK, Duarte FM, Sankar V, Chiang Z, Brack A, Law T, Hauck H, Okimoto A, Regev A, Buenrostro JD, Jacks T. 2020. Epigenomic State Transitions Characterize Tumor Progression in Mouse Lung Adenocarcinoma. Cancer cell. 38(2):212-228.e13. Pubmed: 32707078 DOI:S1535-6108(20)30310-X LaFave LM, Kartha VK, Ma S, Meli K, Del Priore I, Lareau C, Naranjo S, Westcott PMK, Duarte FM, Sankar V, Chiang Z, Brack A, Law T, Hauck H, Okimoto A, Regev A, Buenrostro JD, Jacks T. 2020. Epigenomic State Transitions Characterize Tumor Progression in Mouse Lung Adenocarcinoma. Cancer cell. 38(2):212-228.e13. Pubmed: 32707078 DOI:S1535-6108(20)30310-X Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression.Copyright © 2020 Elsevier Inc. All rights reserved. -
Sarikhani M, Garbern JC, Ma S, Sereda R, Conde J, Krähenbühl G, Escalante GO, Ahmed A, Buenrostro JD, Lee RT. 2020. Sustained Activation of AMPK Enhances Differentiation of Human iPSC-Derived Cardiomyocytes via Sirtuin Activation. Stem cell reports. 15(2):498-514. Pubmed: 32649901 DOI:S2213-6711(20)30233-2 Sarikhani M, Garbern JC, Ma S, Sereda R, Conde J, Krähenbühl G, Escalante GO, Ahmed A, Buenrostro JD, Lee RT. 2020. Sustained Activation of AMPK Enhances Differentiation of Human iPSC-Derived Cardiomyocytes via Sirtuin Activation. Stem cell reports. 15(2):498-514. Pubmed: 32649901 DOI:S2213-6711(20)30233-2 Recent studies suggest that metabolic regulation may improve differentiation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs). AMP-activated protein kinase (AMPK) is a master regulator of metabolic activities. We investigated whether AMPK participates in iPSC-derived cardiomyocyte differentiation. We observed that AMPK phosphorylation at Thr172 increased at day 9 but then decreased after day 11 of differentiation to cardiomyocytes. Inhibition of AMPK with compound C significantly reduced mRNA and protein expression of cardiac troponins TNNT2 and TNNI3. Moreover, sustained AMPK activation using AICAR from days 9 to 14 of differentiation increased mRNA and protein expression of both TNNT2 and TNNI3. AICAR decreased acetylation of histone 3 at Lys9 and 56 and histone 4 at Lys16 (known target sites for nuclear-localized sirtuins [SIRT1, SIRT6]), suggesting that AMPK activation enhances sirtuin activity. Sustained AMPK activation during days 9-14 of differentiation induces sirtuin-mediated histone deacetylation and may enhance cardiomyocyte differentiation from iPSCs.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved. -
Lareau CA, Ma S, Duarte FM, Buenrostro JD. 2020. Inference and effects of barcode multiplets in droplet-based single-cell assays. Nature communications. 11(1):866. Pubmed: 32054859 DOI:10.1038/s41467-020-14667-5 Lareau CA, Ma S, Duarte FM, Buenrostro JD. 2020. Inference and effects of barcode multiplets in droplet-based single-cell assays. Nature communications. 11(1):866. Pubmed: 32054859 DOI:10.1038/s41467-020-14667-5 A widespread assumption for single-cell analyses specifies that one cell's nucleic acids are predominantly captured by one oligonucleotide barcode. Here, we show that ~13-21% of cell barcodes from the 10x Chromium scATAC-seq assay may have been derived from a droplet with more than one oligonucleotide sequence, which we call "barcode multiplets". We demonstrate that barcode multiplets can be derived from at least two different sources. First, we confirm that approximately 4% of droplets from the 10x platform may contain multiple beads. Additionally, we find that approximately 5% of beads may contain detectable levels of multiple oligonucleotide barcodes. We show that this artifact can confound single-cell analyses, including the interpretation of clonal diversity and proliferation of intra-tumor lymphocytes. Overall, our work provides a conceptual and computational framework to identify and assess the impacts of barcode multiplets in single-cell data. -
Zhang B, Ma S, Rachmin I, He M, Baral P, Choi S, Gonçalves WA, Shwartz Y, Fast EM, Su Y, Zon LI, Regev A, Buenrostro JD, Cunha TM, Chiu IM, Fisher DE, Hsu YC. 2020. Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells. Nature. 577(7792):676-681. Pubmed: 31969699 DOI:10.1038/s41586-020-1935-3 Zhang B, Ma S, Rachmin I, He M, Baral P, Choi S, Gonçalves WA, Shwartz Y, Fast EM, Su Y, Zon LI, Regev A, Buenrostro JD, Cunha TM, Chiu IM, Fisher DE, Hsu YC. 2020. Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells. Nature. 577(7792):676-681. Pubmed: 31969699 DOI:10.1038/s41586-020-1935-3 Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs), but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism. 2019
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Chen H, Lareau C, Andreani T, Vinyard ME, Garcia SP, Clement K, Andrade-Navarro MA, Buenrostro JD, Pinello L. 2019. Assessment of computational methods for the analysis of single-cell ATAC-seq data. Genome biology. 20(1):241. Pubmed: 31739806 DOI:10.1186/s13059-019-1854-5 Chen H, Lareau C, Andreani T, Vinyard ME, Garcia SP, Clement K, Andrade-Navarro MA, Buenrostro JD, Pinello L. 2019. Assessment of computational methods for the analysis of single-cell ATAC-seq data. Genome biology. 20(1):241. Pubmed: 31739806 DOI:10.1186/s13059-019-1854-5 Array -
Lareau CA, Duarte FM, Chew JG, Kartha VK, Burkett ZD, Kohlway AS, Pokholok D, Aryee MJ, Steemers FJ, Lebofsky R, Buenrostro JD. 2019. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature biotechnology. 37(8):916-924. Pubmed: 31235917 DOI:10.1038/s41587-019-0147-6 Lareau CA, Duarte FM, Chew JG, Kartha VK, Burkett ZD, Kohlway AS, Pokholok D, Aryee MJ, Steemers FJ, Lebofsky R, Buenrostro JD. 2019. Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature biotechnology. 37(8):916-924. Pubmed: 31235917 DOI:10.1038/s41587-019-0147-6 Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (10 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform. -
Ludwig LS, Lareau CA, Bao EL, Nandakumar SK, Muus C, Ulirsch JC, Chowdhary K, Buenrostro JD, Mohandas N, An X, Aryee MJ, Regev A, Sankaran VG. 2019. Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis. Cell reports. 27(11):3228-3240.e7. Pubmed: 31189107 DOI:S2211-1247(19)30666-7 Ludwig LS, Lareau CA, Bao EL, Nandakumar SK, Muus C, Ulirsch JC, Chowdhary K, Buenrostro JD, Mohandas N, An X, Aryee MJ, Regev A, Sankaran VG. 2019. Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis. Cell reports. 27(11):3228-3240.e7. Pubmed: 31189107 DOI:S2211-1247(19)30666-7 Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved. -
Yoshida H, Lareau CA, Ramirez RN, Rose SA, Maier B, Wroblewska A, Desland F, Chudnovskiy A, Mortha A, Dominguez C, Tellier J, Kim E, Dwyer D, Shinton S, Nabekura T, Qi Y, Yu B, Robinette M, Kim KW, Wagers A, Rhoads A, Nutt SL, Brown BD, Mostafavi S, Buenrostro JD, Benoist C. 2019. The cis-Regulatory Atlas of the Mouse Immune System. Cell. 176(4):897-912.e20. Pubmed: 30686579 DOI:S0092-8674(18)31650-7 Yoshida H, Lareau CA, Ramirez RN, Rose SA, Maier B, Wroblewska A, Desland F, Chudnovskiy A, Mortha A, Dominguez C, Tellier J, Kim E, Dwyer D, Shinton S, Nabekura T, Qi Y, Yu B, Robinette M, Kim KW, Wagers A, Rhoads A, Nutt SL, Brown BD, Mostafavi S, Buenrostro JD, Benoist C. 2019. The cis-Regulatory Atlas of the Mouse Immune System. Cell. 176(4):897-912.e20. Pubmed: 30686579 DOI:S0092-8674(18)31650-7 A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.Copyright © 2018 Elsevier Inc. All rights reserved. -
Shema E, Bernstein BE, Buenrostro JD. 2019. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nature genetics. 51(1):19-25. Pubmed: 30559489 DOI:10.1038/s41588-018-0290-x Shema E, Bernstein BE, Buenrostro JD. 2019. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nature genetics. 51(1):19-25. Pubmed: 30559489 DOI:10.1038/s41588-018-0290-x Recent advances in single-cell and single-molecule epigenomic technologies now enable the study of genome regulation and dynamics at unprecedented resolution. In this Perspective, we highlight some of these transformative technologies and discuss how they have been used to identify new modes of gene regulation. We also contrast these assays with recent advances in single-cell transcriptomics and argue for the essential role of epigenomic technologies in both understanding cellular diversity and discovering gene regulatory mechanisms. In addition, we provide our view on the next generation of biological tools that we expect will open new avenues for elucidating the fundamental principles of gene regulation. Overall, this Perspective motivates the use of these high-resolution epigenomic technologies for mapping cell states and understanding regulatory diversity at single-molecule resolution within single cells. -
Ludwig LS, Lareau CA, Ulirsch JC, Christian E, Muus C, Li LH, Pelka K, Ge W, Oren Y, Brack A, Law T, Rodman C, Chen JH, Boland GM, Hacohen N, Rozenblatt-Rosen O, Aryee MJ, Buenrostro JD, Regev A, Sankaran VG. 2019. Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics. Cell. 176(6):1325-1339.e22. Pubmed: 30827679 DOI:S0092-8674(19)30055-8 Ludwig LS, Lareau CA, Ulirsch JC, Christian E, Muus C, Li LH, Pelka K, Ge W, Oren Y, Brack A, Law T, Rodman C, Chen JH, Boland GM, Hacohen N, Rozenblatt-Rosen O, Aryee MJ, Buenrostro JD, Regev A, Sankaran VG. 2019. Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics. Cell. 176(6):1325-1339.e22. Pubmed: 30827679 DOI:S0092-8674(19)30055-8 Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.Copyright © 2019 Elsevier Inc. All rights reserved. -
Ulirsch JC, Lareau CA, Bao EL, Ludwig LS, Guo MH, Benner C, Satpathy AT, Kartha VK, Salem RM, Hirschhorn JN, Finucane HK, Aryee MJ, Buenrostro JD, Sankaran VG. 2019. Interrogation of human hematopoiesis at single-cell and single-variant resolution. Nature genetics. 51(4):683-693. Pubmed: 30858613 DOI:10.1038/s41588-019-0362-6 Ulirsch JC, Lareau CA, Bao EL, Ludwig LS, Guo MH, Benner C, Satpathy AT, Kartha VK, Salem RM, Hirschhorn JN, Finucane HK, Aryee MJ, Buenrostro JD, Sankaran VG. 2019. Interrogation of human hematopoiesis at single-cell and single-variant resolution. Nature genetics. 51(4):683-693. Pubmed: 30858613 DOI:10.1038/s41588-019-0362-6 Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations. -
Zviran A, Mor N, Rais Y, Gingold H, Peles S, Chomsky E, Viukov S, Buenrostro JD, Scognamiglio R, Weinberger L, Manor YS, Krupalnik V, Zerbib M, Hezroni H, Jaitin DA, Larastiaso D, Gilad S, Benjamin S, Gafni O, Mousa A, Ayyash M, Sheban D, Bayerl J, Aguilera-Castrejon A, Massarwa R, Maza I, Hanna S, Stelzer Y, Ulitsky I, Greenleaf WJ, Tanay A, Trumpp A, Amit I, Pilpel Y, Novershtern N, Hanna JH. 2019. Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules. Cell stem cell. 24(2):328-341.e9. Pubmed: 30554962 DOI:S1934-5909(18)30550-2 Zviran A, Mor N, Rais Y, Gingold H, Peles S, Chomsky E, Viukov S, Buenrostro JD, Scognamiglio R, Weinberger L, Manor YS, Krupalnik V, Zerbib M, Hezroni H, Jaitin DA, Larastiaso D, Gilad S, Benjamin S, Gafni O, Mousa A, Ayyash M, Sheban D, Bayerl J, Aguilera-Castrejon A, Massarwa R, Maza I, Hanna S, Stelzer Y, Ulitsky I, Greenleaf WJ, Tanay A, Trumpp A, Amit I, Pilpel Y, Novershtern N, Hanna JH. 2019. Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules. Cell stem cell. 24(2):328-341.e9. Pubmed: 30554962 DOI:S1934-5909(18)30550-2 The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.Copyright © 2018 Elsevier Inc. All rights reserved. -
Chen H, Albergante L, Hsu JY, Lareau CA, Lo Bosco G, Guan J, Zhou S, Gorban AN, Bauer DE, Aryee MJ, Langenau DM, Zinovyev A, Buenrostro JD, Yuan GC, Pinello L. 2019. Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM. Nature communications. 10(1):1903. Pubmed: 31015418 DOI:10.1038/s41467-019-09670-4 Chen H, Albergante L, Hsu JY, Lareau CA, Lo Bosco G, Guan J, Zhou S, Gorban AN, Bauer DE, Aryee MJ, Langenau DM, Zinovyev A, Buenrostro JD, Yuan GC, Pinello L. 2019. Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM. Nature communications. 10(1):1903. Pubmed: 31015418 DOI:10.1038/s41467-019-09670-4 Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package. 2018
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Finucane HK, Reshef YA, Anttila V, Slowikowski K, Gusev A, Byrnes A, Gazal S, Loh PR, Lareau C, Shoresh N, Genovese G, Saunders A, Macosko E, Pollack S, Perry JRB, Buenrostro JD, Bernstein BE, Raychaudhuri S, McCarroll S, Neale BM, Price AL. 2018. Heritability enrichment of specifically expressed genes identifies disease-relevant tissues and cell types. Nature genetics. 50(4):621-629. Pubmed: 29632380 DOI:10.1038/s41588-018-0081-4 Finucane HK, Reshef YA, Anttila V, Slowikowski K, Gusev A, Byrnes A, Gazal S, Loh PR, Lareau C, Shoresh N, Genovese G, Saunders A, Macosko E, Pollack S, Perry JRB, Buenrostro JD, Bernstein BE, Raychaudhuri S, McCarroll S, Neale BM, Price AL. 2018. Heritability enrichment of specifically expressed genes identifies disease-relevant tissues and cell types. Nature genetics. 50(4):621-629. Pubmed: 29632380 DOI:10.1038/s41588-018-0081-4 We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals. -
Satpathy AT, Saligrama N, Buenrostro JD, Wei Y, Wu B, Rubin AJ, Granja JM, Lareau CA, Li R, Qi Y, Parker KR, Mumbach MR, Serratelli WS, Gennert DG, Schep AN, Corces MR, Khodadoust MS, Kim YH, Khavari PA, Greenleaf WJ, Davis MM, Chang HY. 2018. Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine. 24(5):580-590. Pubmed: 29686426 DOI:10.1038/s41591-018-0008-8 Satpathy AT, Saligrama N, Buenrostro JD, Wei Y, Wu B, Rubin AJ, Granja JM, Lareau CA, Li R, Qi Y, Parker KR, Mumbach MR, Serratelli WS, Gennert DG, Schep AN, Corces MR, Khodadoust MS, Kim YH, Khavari PA, Greenleaf WJ, Davis MM, Chang HY. 2018. Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine. 24(5):580-590. Pubmed: 29686426 DOI:10.1038/s41591-018-0008-8 T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4 T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy. -
Buenrostro JD, Corces MR, Lareau CA, Wu B, Schep AN, Aryee MJ, Majeti R, Chang HY, Greenleaf WJ. 2018. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell. 173(6):1535-1548.e16. Pubmed: 29706549 DOI:S0092-8674(18)30446-X Buenrostro JD, Corces MR, Lareau CA, Wu B, Schep AN, Aryee MJ, Majeti R, Chang HY, Greenleaf WJ. 2018. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell. 173(6):1535-1548.e16. Pubmed: 29706549 DOI:S0092-8674(18)30446-X Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.Copyright © 2018 Elsevier Inc. All rights reserved. -
Chihara N, Madi A, Kondo T, Zhang H, Acharya N, Singer M, Nyman J, Marjanovic ND, Kowalczyk MS, Wang C, Kurtulus S, Law T, Etminan Y, Nevin J, Buckley CD, Burkett PR, Buenrostro JD, Rozenblatt-Rosen O, Anderson AC, Regev A, Kuchroo VK. 2018. Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature. 558(7710):454-459. Pubmed: 29899446 DOI:10.1038/s41586-018-0206-z Chihara N, Madi A, Kondo T, Zhang H, Acharya N, Singer M, Nyman J, Marjanovic ND, Kowalczyk MS, Wang C, Kurtulus S, Law T, Etminan Y, Nevin J, Buckley CD, Burkett PR, Buenrostro JD, Rozenblatt-Rosen O, Anderson AC, Regev A, Kuchroo VK. 2018. Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature. 558(7710):454-459. Pubmed: 29899446 DOI:10.1038/s41586-018-0206-z The expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated expression of co-inhibitory receptors on CD4 T cells promotes autoimmunity, whereas sustained overexpression on CD8 T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and diseases such as cancer. Here, using RNA and protein expression profiling at single-cell resolution in mouse cells, we identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, TIM-3, LAG-3 and TIGIT) but also many new surface receptors. We functionally validated two new co-inhibitory receptors, activated protein C receptor (PROCR) and podoplanin (PDPN). The module of co-inhibitory receptors is co-expressed in both CD4 and CD8 T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in several physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors PRDM1 and c-MAF as cooperative regulators of the co-inhibitory module, and this was validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies regulators of T cell function with the potential to control autoimmunity and tumour immunity. -
Mor N, Rais Y, Sheban D, Peles S, Aguilera-Castrejon A, Zviran A, Elinger D, Viukov S, Geula S, Krupalnik V, Zerbib M, Chomsky E, Lasman L, Shani T, Bayerl J, Gafni O, Hanna S, Buenrostro JD, Hagai T, Masika H, Vainorius G, Bergman Y, Greenleaf WJ, Esteban MA, Elling U, Levin Y, Massarwa R, Merbl Y, Novershtern N, Hanna JH. 2018. Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency. Cell stem cell. 23(3):412-425.e10. Pubmed: 30122475 DOI:S1934-5909(18)30340-0 Mor N, Rais Y, Sheban D, Peles S, Aguilera-Castrejon A, Zviran A, Elinger D, Viukov S, Geula S, Krupalnik V, Zerbib M, Chomsky E, Lasman L, Shani T, Bayerl J, Gafni O, Hanna S, Buenrostro JD, Hagai T, Masika H, Vainorius G, Bergman Y, Greenleaf WJ, Esteban MA, Elling U, Levin Y, Massarwa R, Merbl Y, Novershtern N, Hanna JH. 2018. Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency. Cell stem cell. 23(3):412-425.e10. Pubmed: 30122475 DOI:S1934-5909(18)30340-0 Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.Copyright © 2018 Elsevier Inc. All rights reserved. -
Koh AS, Miller EL, Buenrostro JD, Moskowitz DM, Wang J, Greenleaf WJ, Chang HY, Crabtree GR. 2018. Rapid chromatin repression by Aire provides precise control of immune tolerance. Nature immunology. 19(2):162-172. Pubmed: 29335648 DOI:10.1038/s41590-017-0032-8 Koh AS, Miller EL, Buenrostro JD, Moskowitz DM, Wang J, Greenleaf WJ, Chang HY, Crabtree GR. 2018. Rapid chromatin repression by Aire provides precise control of immune tolerance. Nature immunology. 19(2):162-172. Pubmed: 29335648 DOI:10.1038/s41590-017-0032-8 Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance. 2017
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Soto-Feliciano YM, Bartlebaugh JME, Liu Y, Sánchez-Rivera FJ, Bhutkar A, Weintraub AS, Buenrostro JD, Cheng CS, Regev A, Jacks TE, Young RA, Hemann MT. 2017. PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes. Genes & development. 31(10):973-989. Pubmed: 28607179 DOI:10.1101/gad.295857.117 Soto-Feliciano YM, Bartlebaugh JME, Liu Y, Sánchez-Rivera FJ, Bhutkar A, Weintraub AS, Buenrostro JD, Cheng CS, Regev A, Jacks TE, Young RA, Hemann MT. 2017. PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes. Genes & development. 31(10):973-989. Pubmed: 28607179 DOI:10.1101/gad.295857.117 Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition.© 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press. -
Guo MH, Nandakumar SK, Ulirsch JC, Zekavat SM, Buenrostro JD, Natarajan P, Salem RM, Chiarle R, Mitt M, Kals M, Pärn K, Fischer K, Milani L, Mägi R, Palta P, Gabriel SB, Metspalu A, Lander ES, Kathiresan S, Hirschhorn JN, Esko T, Sankaran VG. 2017. Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 114(3):E327-E336. Pubmed: 28031487 DOI:10.1073/pnas.1619052114 Guo MH, Nandakumar SK, Ulirsch JC, Zekavat SM, Buenrostro JD, Natarajan P, Salem RM, Chiarle R, Mitt M, Kals M, Pärn K, Fischer K, Milani L, Mägi R, Palta P, Gabriel SB, Metspalu A, Lander ES, Kathiresan S, Hirschhorn JN, Esko T, Sankaran VG. 2017. Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 114(3):E327-E336. Pubmed: 28031487 DOI:10.1073/pnas.1619052114 Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance. -
Litzenburger UM, Buenrostro JD, Wu B, Shen Y, Sheffield NC, Kathiria A, Greenleaf WJ, Chang HY. 2017. Single-cell epigenomic variability reveals functional cancer heterogeneity. Genome biology. 18(1):15. Pubmed: 28118844 DOI:10.1186/s13059-016-1133-7 Litzenburger UM, Buenrostro JD, Wu B, Shen Y, Sheffield NC, Kathiria A, Greenleaf WJ, Chang HY. 2017. Single-cell epigenomic variability reveals functional cancer heterogeneity. Genome biology. 18(1):15. Pubmed: 28118844 DOI:10.1186/s13059-016-1133-7 Array -
Moskowitz DM, Zhang DW, Hu B, Le Saux S, Yanes RE, Ye Z, Buenrostro JD, Weyand CM, Greenleaf WJ, Goronzy JJ. 2017. Epigenomics of human CD8 T cell differentiation and aging. Science immunology. 2(8). Pubmed: 28439570 DOI:10.1126/sciimmunol.aag0192 Moskowitz DM, Zhang DW, Hu B, Le Saux S, Yanes RE, Ye Z, Buenrostro JD, Weyand CM, Greenleaf WJ, Goronzy JJ. 2017. Epigenomics of human CD8 T cell differentiation and aging. Science immunology. 2(8). Pubmed: 28439570 DOI:10.1126/sciimmunol.aag0192 The efficacy of the adaptive immune response declines dramatically with age, but the cell-intrinsic mechanisms driving immune aging in humans remain poorly understood. Immune aging is characterized by a loss of self-renewing naïve cells and the accumulation of differentiated but dysfunctional cells within the CD8 T cell compartment. Using ATAC-seq, we inferred the transcription factor binding activities correlated with naive and central and effector memory CD8 T cell states in young adults. Integrating our results with RNA-seq, we identified transcription networks associated with CD8 T cell differentiation, with prominent roles implicated for BATF, ETS1, Eomes, and Sp1. Extending our analysis to aged humans, we found that the differences between the memory and naive subsets were largely preserved across age, but that naive and central memory cells from older individuals exhibited a shift toward more differentiated patterns of chromatin openness. Additionally, aged naive cells displayed a loss in chromatin accessibility at gene promoters, largely associated with a decrease in NRF1 binding. This shift was implicated in a marked drop-off in the ability of the aged naive cells to transcribe respiratory chain genes, which may explain the reduced capacity of oxidative phosphorylation in older naïve cells. Our findings identify BATF- and NRF1-driven gene regulation as potential targets for delaying CD8 T cell aging and restoring function. -
Miller EL, Hargreaves DC, Kadoch C, Chang CY, Calarco JP, Hodges C, Buenrostro JD, Cui K, Greenleaf WJ, Zhao K, Crabtree GR. 2017. TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin. Nature structural & molecular biology. 24(4):344-352. Pubmed: 28250416 DOI:10.1038/nsmb.3384 Miller EL, Hargreaves DC, Kadoch C, Chang CY, Calarco JP, Hodges C, Buenrostro JD, Cui K, Greenleaf WJ, Zhao K, Crabtree GR. 2017. TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin. Nature structural & molecular biology. 24(4):344-352. Pubmed: 28250416 DOI:10.1038/nsmb.3384 The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures. -
Daugherty AC, Yeo RW, Buenrostro JD, Greenleaf WJ, Kundaje A, Brunet A. 2017. Chromatin accessibility dynamics reveal novel functional enhancers in. Genome research. 27(12):2096-2107. Pubmed: 29141961 DOI:10.1101/gr.226233.117 Daugherty AC, Yeo RW, Buenrostro JD, Greenleaf WJ, Kundaje A, Brunet A. 2017. Chromatin accessibility dynamics reveal novel functional enhancers in. Genome research. 27(12):2096-2107. Pubmed: 29141961 DOI:10.1101/gr.226233.117 Chromatin accessibility, a crucial component of genome regulation, has primarily been studied in homogeneous and simple systems, such as isolated cell populations or early-development models. Whether chromatin accessibility can be assessed in complex, dynamic systems in vivo with high sensitivity remains largely unexplored. In this study, we use ATAC-seq to identify chromatin accessibility changes in a whole animal, the model organism , from embryogenesis to adulthood. Chromatin accessibility changes between developmental stages are highly reproducible, recapitulate histone modification changes, and reveal key regulatory aspects of the epigenomic landscape throughout organismal development. We find that over 5000 distal noncoding regions exhibit dynamic changes in chromatin accessibility between developmental stages and could thereby represent putative enhancers. When tested in vivo, several of these putative enhancers indeed drive novel cell-type- and temporal-specific patterns of expression. Finally, by integrating transcription factor binding motifs in a machine learning framework, we identify EOR-1 as a unique transcription factor that may regulate chromatin dynamics during development. Our study provides a unique resource for , a system in which the prevalence and importance of enhancers remains poorly characterized, and demonstrates the power of using whole organism chromatin accessibility to identify novel regulatory regions in complex systems.© 2017 Daugherty et al.; Published by Cold Spring Harbor Laboratory Press. -
Schep AN, Wu B, Buenrostro JD, Greenleaf WJ. 2017. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nature methods. 14(10):975-978. Pubmed: 28825706 DOI:10.1038/nmeth.4401 Schep AN, Wu B, Buenrostro JD, Greenleaf WJ. 2017. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nature methods. 14(10):975-978. Pubmed: 28825706 DOI:10.1038/nmeth.4401 Single-cell ATAC-seq (scATAC) yields sparse data that make conventional analysis challenging. We developed chromVAR (http://www.github.com/GreenleafLab/chromVAR), an R package for analyzing sparse chromatin-accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and characterization of known and de novo sequence motifs associated with variation in chromatin accessibility. -
She R, Chakravarty AK, Layton CJ, Chircus LM, Andreasson JO, Damaraju N, McMahon PL, Buenrostro JD, Jarosz DF, Greenleaf WJ. 2017. Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome. Proceedings of the National Academy of Sciences of the United States of America. 114(14):3619-3624. Pubmed: 28325876 DOI:10.1073/pnas.1618370114 She R, Chakravarty AK, Layton CJ, Chircus LM, Andreasson JO, Damaraju N, McMahon PL, Buenrostro JD, Jarosz DF, Greenleaf WJ. 2017. Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome. Proceedings of the National Academy of Sciences of the United States of America. 114(14):3619-3624. Pubmed: 28325876 DOI:10.1073/pnas.1618370114 RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP. 2016
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Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, Snyder MP, Pritchard JK, Kundaje A, Greenleaf WJ, Majeti R, Chang HY. 2016. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics. 48(10):1193-203. Pubmed: 27526324 DOI:10.1038/ng.3646 Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, Snyder MP, Pritchard JK, Kundaje A, Greenleaf WJ, Majeti R, Chang HY. 2016. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics. 48(10):1193-203. Pubmed: 27526324 DOI:10.1038/ng.3646 We define the chromatin accessibility and transcriptional landscapes in 13 human primary blood cell types that span the hematopoietic hierarchy. Exploiting the finding that the enhancer landscape better reflects cell identity than mRNA levels, we enable 'enhancer cytometry' for enumeration of pure cell types from complex populations. We identify regulators governing hematopoietic differentiation and further show the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia (AML), chromatin accessibility uncovers unique regulatory evolution in cancer cells with a progressively increasing mutation burden. Single AML cells exhibit distinctive mixed regulome profiles corresponding to disparate developmental stages. A method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of preleukemic hematopoietic stem cell characteristics. Thus, regulome dynamics can provide diverse insights into hematopoietic development and disease. -
Chen X, Shen Y, Draper W, Buenrostro JD, Litzenburger U, Cho SW, Satpathy AT, Carter AC, Ghosh RP, East-Seletsky A, Doudna JA, Greenleaf WJ, Liphardt JT, Chang HY. 2016. ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nature methods. 13(12):1013-1020. Pubmed: 27749837 DOI:10.1038/nmeth.4031 Chen X, Shen Y, Draper W, Buenrostro JD, Litzenburger U, Cho SW, Satpathy AT, Carter AC, Ghosh RP, East-Seletsky A, Doudna JA, Greenleaf WJ, Liphardt JT, Chang HY. 2016. ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nature methods. 13(12):1013-1020. Pubmed: 27749837 DOI:10.1038/nmeth.4031 Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. 2015
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Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. 2015. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Current protocols in molecular biology. 109:21.29.1-21.29.9. Pubmed: 25559105 DOI:10.1002/0471142727.mb2129s109 Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. 2015. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Current protocols in molecular biology. 109:21.29.1-21.29.9. Pubmed: 25559105 DOI:10.1002/0471142727.mb2129s109 This unit describes Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), a method for mapping chromatin accessibility genome-wide. This method probes DNA accessibility with hyperactive Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin. Sequencing reads can then be used to infer regions of increased accessibility, as well as to map regions of transcription-factor binding and nucleosome position. The method is a fast and sensitive alternative to DNase-seq for assaying chromatin accessibility genome-wide, or to MNase-seq for assaying nucleosome positions in accessible regions of the genome.Copyright © 2015 John Wiley & Sons, Inc. -
Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. 2015. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 523(7561):486-90. Pubmed: 26083756 DOI:10.1038/nature14590 Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. 2015. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 523(7561):486-90. Pubmed: 26083756 DOI:10.1038/nature14590 Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'. -
Maza I, Caspi I, Zviran A, Chomsky E, Rais Y, Viukov S, Geula S, Buenrostro JD, Weinberger L, Krupalnik V, Hanna S, Zerbib M, Dutton JR, Greenleaf WJ, Massarwa R, Novershtern N, Hanna JH. 2015. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nature biotechnology. 33(7):769-74. Pubmed: 26098448 DOI:10.1038/nbt.3270 Maza I, Caspi I, Zviran A, Chomsky E, Rais Y, Viukov S, Geula S, Buenrostro JD, Weinberger L, Krupalnik V, Hanna S, Zerbib M, Dutton JR, Greenleaf WJ, Massarwa R, Novershtern N, Hanna JH. 2015. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nature biotechnology. 33(7):769-74. Pubmed: 26098448 DOI:10.1038/nbt.3270 Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods. -
Schep AN, Buenrostro JD, Denny SK, Schwartz K, Sherlock G, Greenleaf WJ. 2015. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions. Genome research. 25(11):1757-70. Pubmed: 26314830 DOI:10.1101/gr.192294.115 Schep AN, Buenrostro JD, Denny SK, Schwartz K, Sherlock G, Greenleaf WJ. 2015. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions. Genome research. 25(11):1757-70. Pubmed: 26314830 DOI:10.1101/gr.192294.115 Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.© 2015 Schep et al.; Published by Cold Spring Harbor Laboratory Press. -
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. 2014
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Buenrostro JD, Araya CL, Chircus LM, Layton CJ, Chang HY, Snyder MP, Greenleaf WJ. 2014. Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nature biotechnology. 32(6):562-8. Pubmed: 24727714 DOI:10.1038/nbt.2880 Buenrostro JD, Araya CL, Chircus LM, Layton CJ, Chang HY, Snyder MP, Greenleaf WJ. 2014. Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nature biotechnology. 32(6):562-8. Pubmed: 24727714 DOI:10.1038/nbt.2880 RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships. -
Couthouis J, Raphael AR, Siskind C, Findlay AR, Buenrostro JD, Greenleaf WJ, Vogel H, Day JW, Flanigan KM, Gitler AD. 2014. Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy. Neuromuscular disorders : NMD. 24(5):431-5. Pubmed: 24594375 DOI:S0960-8966(14)00047-9 Couthouis J, Raphael AR, Siskind C, Findlay AR, Buenrostro JD, Greenleaf WJ, Vogel H, Day JW, Flanigan KM, Gitler AD. 2014. Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy. Neuromuscular disorders : NMD. 24(5):431-5. Pubmed: 24594375 DOI:S0960-8966(14)00047-9 Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D.Copyright © 2014 Elsevier B.V. All rights reserved. 2013
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Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods. 10(12):1213-8. Pubmed: 24097267 DOI:10.1038/nmeth.2688 Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods. 10(12):1213-8. Pubmed: 24097267 DOI:10.1038/nmeth.2688 We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making. -
Carpenter ML, Buenrostro JD, Valdiosera C, Schroeder H, Allentoft ME, Sikora M, Rasmussen M, Gravel S, Guillén S, Nekhrizov G, Leshtakov K, Dimitrova D, Theodossiev N, Pettener D, Luiselli D, Sandoval K, Moreno-Estrada A, Li Y, Wang J, Gilbert MT, Willerslev E, Greenleaf WJ, Bustamante CD. 2013. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries. American journal of human genetics. 93(5):852-64. Pubmed: 24568772 DOI:S0002-9297(13)00459-X Carpenter ML, Buenrostro JD, Valdiosera C, Schroeder H, Allentoft ME, Sikora M, Rasmussen M, Gravel S, Guillén S, Nekhrizov G, Leshtakov K, Dimitrova D, Theodossiev N, Pettener D, Luiselli D, Sandoval K, Moreno-Estrada A, Li Y, Wang J, Gilbert MT, Willerslev E, Greenleaf WJ, Bustamante CD. 2013. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries. American journal of human genetics. 93(5):852-64. Pubmed: 24568772 DOI:S0002-9297(13)00459-X Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved. 2012
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Flaherty P, Natsoulis G, Muralidharan O, Winters M, Buenrostro J, Bell J, Brown S, Holodniy M, Zhang N, Ji HP. 2012. Ultrasensitive detection of rare mutations using next-generation targeted resequencing. Nucleic acids research. 40(1):e2. Pubmed: 22013163 DOI:10.1093/nar/gkr861 Flaherty P, Natsoulis G, Muralidharan O, Winters M, Buenrostro J, Bell J, Brown S, Holodniy M, Zhang N, Ji HP. 2012. Ultrasensitive detection of rare mutations using next-generation targeted resequencing. Nucleic acids research. 40(1):e2. Pubmed: 22013163 DOI:10.1093/nar/gkr861 With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection levels are limited by the error rate of the sequencing technology as well as by the availability of variant-calling algorithms with high statistical power and low false positive rates. We demonstrate that we can robustly detect mutations at 0.1% fractional representation. This represents accurate detection of one mutant per every 1000 wild-type alleles. To achieve this sensitive level of mutation detection, we integrate a high accuracy indexing strategy and reference replication for estimating sequencing error variance. We employ a statistical model to estimate the error rate at each position of the reference and to quantify the fraction of variant base in the sample. Our method is highly specific (99%) and sensitive (100%) when applied to a known 0.1% sample fraction admixture of two synthetic DNA samples to validate our method. As a clinical application of this method, we analyzed nine clinical samples of H1N1 influenza A and detected an oseltamivir (antiviral therapy) resistance mutation in the H1N1 neuraminidase gene at a sample fraction of 0.18%. 2011
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Dick CA, Buenrostro J, Butler T, Carlson ML, Kliebenstein DJ, Whittall JB. 2011. Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway. PloS one. 6(4):e18230. Pubmed: 21490971 DOI:10.1371/journal.pone.0018230 Dick CA, Buenrostro J, Butler T, Carlson ML, Kliebenstein DJ, Whittall JB. 2011. Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway. PloS one. 6(4):e18230. Pubmed: 21490971 DOI:10.1371/journal.pone.0018230 Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s) of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP), suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS) at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments. -
Natsoulis G, Bell JM, Xu H, Buenrostro JD, Ordonez H, Grimes S, Newburger D, Jensen M, Zahn JM, Zhang N, Ji HP. 2011. A flexible approach for highly multiplexed candidate gene targeted resequencing. PloS one. 6(6):e21088. Pubmed: 21738606 DOI:10.1371/journal.pone.0021088 Natsoulis G, Bell JM, Xu H, Buenrostro JD, Ordonez H, Grimes S, Newburger D, Jensen M, Zahn JM, Zhang N, Ji HP. 2011. A flexible approach for highly multiplexed candidate gene targeted resequencing. PloS one. 6(6):e21088. Pubmed: 21738606 DOI:10.1371/journal.pone.0021088 We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies. -
Myllykangas S, Buenrostro JD, Natsoulis G, Bell JM, Ji HP. 2011. Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing. Nature biotechnology. 29(11):1024-7. Pubmed: 22020387 DOI:10.1038/nbt.1996 Myllykangas S, Buenrostro JD, Natsoulis G, Bell JM, Ji HP. 2011. Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing. Nature biotechnology. 29(11):1024-7. Pubmed: 22020387 DOI:10.1038/nbt.1996 We describe an approach for targeted genome resequencing, called oligonucleotide-selective sequencing (OS-Seq), in which we modify the immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer to function as both a capture and sequencing substrate. We apply OS-Seq to resequence the exons of either 10 or 344 cancer genes from human DNA samples. In our assessment of capture performance, >87% of the captured sequence originated from the intended target region with sequencing coverage falling within a tenfold range for a majority of all targets. Single nucleotide variants (SNVs) called from OS-Seq data agreed with >95% of variants obtained from whole-genome sequencing of the same individual. We also demonstrate mutation discovery from a colorectal cancer tumor sample matched with normal tissue. Overall, we show the robust performance and utility of OS-Seq for the resequencing analysis of human germline and cancer genomes. 2010
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Whittall JB, Syring J, Parks M, Buenrostro J, Dick C, Liston A, Cronn R. 2010. Finding a (pine) needle in a haystack: chloroplast genome sequence divergence in rare and widespread pines. Molecular ecology. 19 Suppl 1:100-14. Pubmed: 20331774 DOI:10.1111/j.1365-294X.2009.04474.x Whittall JB, Syring J, Parks M, Buenrostro J, Dick C, Liston A, Cronn R. 2010. Finding a (pine) needle in a haystack: chloroplast genome sequence divergence in rare and widespread pines. Molecular ecology. 19 Suppl 1:100-14. Pubmed: 20331774 DOI:10.1111/j.1365-294X.2009.04474.x Critical to conservation efforts and other investigations at low taxonomic levels, DNA sequence data offer important insights into the distinctiveness, biogeographic partitioning and evolutionary histories of species. The resolving power of DNA sequences is often limited by insufficient variability at the intraspecific level. This is particularly true of studies involving plant organelles, as the conservative mutation rate of chloroplasts and mitochondria makes it difficult to detect polymorphisms necessary to track genealogical relationships among individuals, populations and closely related taxa, through space and time. Massively parallel sequencing (MPS) makes it possible to acquire entire organelle genome sequences to identify cryptic variation that would be difficult to detect otherwise. We are using MPS to evaluate intraspecific chloroplast-level divergence across biogeographic boundaries in narrowly endemic and widespread species of Pinus. We focus on one of the world's rarest pines - Torrey pine (Pinus torreyana) - due to its conservation interest and because it provides a marked contrast to more widespread pine species. Detailed analysis of nearly 90% ( approximately 105 000 bp each) of these chloroplast genomes shows that mainland and island populations of Torrey pine differ at five sites in their plastome, with the differences fixed between populations. This is an exceptionally low level of divergence (1 polymorphism/ approximately 21 kb), yet it is comparable to intraspecific divergence present in widespread pine species and species complexes. Population-level organelle genome sequencing offers new vistas into the timing and magnitude of divergence within species, and is certain to provide greater insight into pollen dispersal, migration patterns and evolutionary dynamics in plants.