RESUMO
Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Epigenômica , Genômica , Glioblastoma/genética , Glioblastoma/patologia , Análise de Célula Única , Microambiente Tumoral , Heterogeneidade GenéticaRESUMO
Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Animais , Humanos , Cromatina , Montagem e Desmontagem da Cromatina , Proteínas Nucleares/metabolismo , Nucleossomos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , CamundongosRESUMO
Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.
Assuntos
Axônios/fisiologia , Cromatina/química , Cílios , Sinapses , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cílios/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Serotonina/metabolismo , Transdução de Sinais , Sinapses/fisiologiaRESUMO
Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for â¼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems.
Assuntos
Cromatina/metabolismo , Genoma Humano , Análise de Célula Única , Adulto , Análise por Conglomerados , Feto/metabolismo , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Especificidade de Órgãos , Filogenia , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de RiscoRESUMO
Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional/genética , Acetilação , Animais , Sequência de Bases , Segregação de Cromossomos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Genoma , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Masculino , Mamíferos/genética , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Cromossomo X/metabolismo , Zigoto/metabolismoRESUMO
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 â¼4,300 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/metabolismoRESUMO
Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Proteína SMARCB1/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Elementos Facilitadores Genéticos/genética , Feminino , Genoma Humano , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína SMARCB1/química , Proteína SMARCB1/metabolismoRESUMO
The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.
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 , TranscriptomaRESUMO
We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in â¼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and â¼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution.
Assuntos
Cromatina/química , Análise de Célula Única/métodos , Animais , Análise por Conglomerados , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TranscriçãoRESUMO
In early mammalian embryos, it remains unclear how the first cell fate bias is initially triggered and amplified toward cell fate segregation. Here, we report that a long noncoding RNA, LincGET, is transiently and asymmetrically expressed in the nucleus of two- to four-cell mouse embryos. Overexpression of LincGET in one of the two-cell blastomeres biases its progeny predominantly toward the inner cell mass (ICM) fate. Mechanistically, LincGET physically binds to CARM1 and promotes the nuclear localization of CARM1, which can further increase the level of H3 methylation at Arginine 26 (H3R26me), activate ICM-specific gene expression, upregulate transposons, and increase global chromatin accessibility. Simultaneous overexpression of LincGET and depletion of Carm1 no longer biased embryonic fate, indicating that the effect of LincGET in directing ICM lineage depends on CARM1. Thus, our data identify LincGET as one of the earliest known lineage regulators to bias cell fate in mammalian 2-cell embryos.
Assuntos
Blastocisto/metabolismo , Blastômeros/metabolismo , Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , RNA Longo não Codificante/biossíntese , Animais , Blastocisto/citologia , Blastômeros/citologia , Feminino , Histonas/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos ICR , Proteína-Arginina N-Metiltransferases/biossíntese , Proteína-Arginina N-Metiltransferases/genética , RNA Longo não Codificante/genéticaRESUMO
The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.
Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Evolução Molecular , Animais , Sítios de Ligação , Ilhas de CpG , Metilação de DNA , Elementos de DNA Transponíveis/genética , Desoxirribonuclease I/metabolismo , Regulação para Baixo , Desenvolvimento Embrionário , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Zigoto/metabolismoRESUMO
RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação V(D)J , Animais , Linhagem Celular , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese , Sinais Direcionadores de Proteínas , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Histone proteins affect gene expression through multiple mechanisms, including through exchange with histone variants. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. Most notably, widely expressed variants of H2B remain elusive. We applied recently developed antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters, and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Further, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a mechanism linking histone variants to chromatin accessibility, transcriptional regulation, neuronal function, and memory. This work further identifies a widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.
Assuntos
Cromatina , Histonas , Memória de Longo Prazo , Neurônios , Sinapses , Histonas/metabolismo , Histonas/genética , Animais , Cromatina/metabolismo , Cromatina/genética , Memória de Longo Prazo/fisiologia , Neurônios/metabolismo , Camundongos , Sinapses/metabolismo , Sinapses/genética , Regiões Promotoras Genéticas , Camundongos Endogâmicos C57BL , Regulação da Expressão Gênica , Transcrição Gênica , Masculino , HumanosRESUMO
The DNA-binding protein SATB2 is genetically linked to human intelligence. We studied its influence on the three-dimensional (3D) epigenome by mapping chromatin interactions and accessibility in control versus SATB2-deficient cortical neurons. We find that SATB2 affects the chromatin looping between enhancers and promoters of neuronal-activity-regulated genes, thus influencing their expression. It also alters A/B compartments, topologically associating domains, and frequently interacting regions. Genes linked to SATB2-dependent 3D genome changes are implicated in highly specialized neuronal functions and contribute to cognitive ability and risk for neuropsychiatric and neurodevelopmental disorders. Non-coding DNA regions with a SATB2-dependent structure are enriched for common variants associated with educational attainment, intelligence, and schizophrenia. Our data establish SATB2 as a cell-type-specific 3D genome modulator, which operates both independently and in cooperation with CCCTC-binding factor (CTCF) to set up the chromatin landscape of pyramidal neurons for cognitive processes.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neurônios/metabolismo , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Genoma , Cognição , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismoRESUMO
In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Feminino , Camundongos , Masculino , Animais , Inativação do Cromossomo X/genética , Impressão Genômica , Células Germinativas , Epigênese Genética , Embrião de Mamíferos , RNA Longo não Codificante/genética , Cromossomo X/genética , Mamíferos/genéticaRESUMO
The ease and throughput of single-cell genomics have steadily improved, and its current trajectory suggests that surveying single-cell populations will become routine. We discuss the merger of quantitative genetics with single-cell genomics and emphasize how this synergizes with advantages intrinsic to plants. Single-cell population genomics provides increased detection resolution when mapping variants that control molecular traits, including gene expression or chromatin accessibility. Additionally, single-cell population genomics reveals the cell types in which variants act and, when combined with organism-level phenotype measurements, unveils which cellular contexts impact higher-order traits. Emerging technologies, notably multiomics, can facilitate the measurement of both genetic changes and genomic traits in single cells, enabling single-cell genetic experiments. The implementation of single-cell genetics will advance the investigation of the genetic architecture of complex molecular traits and provide new experimental paradigms to study eukaryotic genetics.
Assuntos
Genômica , Herança Multifatorial , Fenótipo , Genoma , Plantas/genéticaRESUMO
How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.
Assuntos
Blastocisto , Cromatina/metabolismo , Animais , Sítios de Ligação , Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Fator de Ligação a CCAAT/metabolismo , Mapeamento Cromossômico , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras GenéticasRESUMO
Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Neoplasias/genética , Sequências Reguladoras de Ácido Nucleico , Regulação da Expressão Gênica , Cromatina/genética , Linfócitos do Interstício Tumoral , Elementos Facilitadores GenéticosRESUMO
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.