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1.
Cell ; 176(1-2): 361-376.e17, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30580963

RESUMO

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.


Assuntos
Epigenômica/métodos , Redes Reguladoras de Genes/genética , Análise de Célula Única/métodos , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Redes Reguladoras de Genes/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo
2.
Cell ; 173(6): 1535-1548.e16, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29706549

RESUMO

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.


Assuntos
Cromatina/química , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Análise de Célula Única , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Epigênese Genética , Epigenômica , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , Células Progenitoras Mieloides/citologia , Análise de Componente Principal , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA , Transcriptoma
3.
Nature ; 523(7561): 486-90, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26083756

RESUMO

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'.


Assuntos
Células/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigenômica , Análise de Célula Única/métodos , Animais , Compartimento Celular , Ciclo Celular/genética , Linhagem Celular , Células/classificação , DNA/genética , DNA/metabolismo , Epigênese Genética , Genoma Humano/genética , Humanos , Microfluídica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transposases/metabolismo
4.
Nat Methods ; 14(10): 975-978, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28825706

RESUMO

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.


Assuntos
Epigenômica/métodos , Análise de Sequência de DNA/métodos , Software , Fatores de Transcrição/metabolismo , Algoritmos , Animais , Linhagem Celular , Regulação da Expressão Gênica
5.
Nat Methods ; 14(10): 959-962, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28846090

RESUMO

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-µm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.


Assuntos
DNA/genética , Congelamento , Genoma , Manejo de Espécimes/métodos , Animais , Encéfalo , Linhagem Celular , Eritrócitos , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Queratinócitos , Camundongos , Replicação de Sequência Autossustentável , Neoplasias da Glândula Tireoide , Transposases/metabolismo
6.
Nature ; 512(7515): 400-5, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25164749

RESUMO

Discovering the structure and dynamics of transcriptional regulatory events in the genome with cellular and temporal resolution is crucial to understanding the regulatory underpinnings of development and disease. We determined the genomic distribution of binding sites for 92 transcription factors and regulatory proteins across multiple stages of Caenorhabditis elegans development by performing 241 ChIP-seq (chromatin immunoprecipitation followed by sequencing) experiments. Integration of regulatory binding and cellular-resolution expression data produced a spatiotemporally resolved metazoan transcription factor binding map. Using this map, we explore developmental regulatory circuits that encode combinatorial logic at the levels of co-binding and co-expression of transcription factors, characterizing the genomic coverage and clustering of regulatory binding, the binding preferences of, and biological processes regulated by, transcription factors, the global transcription factor co-associations and genomic subdomains that suggest shared patterns of regulation, and identifying key transcription factors and transcription factor co-associations for fate specification of individual lineages and cell types.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma Helmíntico/genética , Análise Espaço-Temporal , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem da Célula , Imunoprecipitação da Cromatina , Genômica , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Ligação Proteica
8.
PLoS Genet ; 9(2): e1003325, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23468654

RESUMO

The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Homeodomínio , Longevidade , Envelhecimento/genética , Processamento Alternativo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Mucosa Intestinal/metabolismo , Longevidade/genética , Longevidade/fisiologia , Especificidade de Órgãos , Hormônios Peptídicos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo
9.
Biophys J ; 100(9): 2121-30, 2011 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-21539779

RESUMO

Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the ß-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the ß-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the ß-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Bicamadas Lipídicas/metabolismo , Dobramento de Proteína , Análise Espectral Raman , Triptofano/metabolismo , Raios Ultravioleta , Adsorção , Bicamadas Lipídicas/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectrometria de Fluorescência , Termodinâmica
10.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570894

RESUMO

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Elementos de Resposta/genética , Linfócitos T/imunologia , Desequilíbrio Alélico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Polissacarídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcriptoma
11.
Nat Med ; 24(5): 580-590, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29686426

RESUMO

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.


Assuntos
Cromatina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transposases/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Transformada , Células Clonais , Epigenômica , Humanos , Imunidade , Células Jurkat , Leucemia/imunologia , Leucemia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única
12.
Genome Biol ; 18(1): 15, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28118844

RESUMO

BACKGROUND: Cell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally. RESULTS: We develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity. CONCLUSION: Single-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution.


Assuntos
Epigênese Genética , Epigenômica , Heterogeneidade Genética , Variação Genética , Neoplasias/genética , Análise de Célula Única , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Células K562 , Neoplasias/metabolismo , Motivos de Nucleotídeos , Reprodutibilidade dos Testes , Análise de Célula Única/métodos
13.
Nat Genet ; 48(10): 1193-203, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27526324

RESUMO

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.


Assuntos
Cromatina , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Linhagem da Célula , Células Clonais , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Mielopoese/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA/métodos , Células Tumorais Cultivadas
14.
Curr Protoc Mol Biol ; 109: 21.29.1-21.29.9, 2015 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-25559105

RESUMO

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.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Técnicas Citológicas/métodos , Biologia Molecular/métodos , Coloração e Rotulagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ligação Proteica , Recombinação Genética , Transposases/metabolismo
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