RESUMO
The dynamics of immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infants and young children by analyzing blood samples and weekly nasal swabs collected before, during, and after infection with Omicron and non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, showed no sign of decay for up to 300 days. Infants mounted a robust mucosal immune response characterized by inflammatory cytokines, interferon (IFN) α, and T helper (Th) 17 and neutrophil markers (interleukin [IL]-17, IL-8, and CXCL1). The immune response in blood was characterized by upregulation of activation markers on innate cells, no inflammatory cytokines, but several chemokines and IFNα. The latter correlated with viral load and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell multi-omics. Together, these data provide a snapshot of immunity to infection during the initial weeks and months of life.
Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Criança , Lactente , Humanos , Pré-Escolar , SARS-CoV-2/metabolismo , Multiômica , Citocinas/metabolismo , Interferon-alfa , Imunidade nas MucosasRESUMO
Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.
Assuntos
Neurogênese , Organoides , Gravidez , Feminino , Humanos , Adulto , Cromatina , Córtex Pré-Frontal , Análise de Célula Única , Redes Reguladoras de GenesRESUMO
cis-regulatory elements (CREs) encode the genomic blueprints of spatiotemporal gene expression programs enabling highly specialized cell functions. Using single-cell genomics in six maize organs, we determined the cis- and trans-regulatory factors defining diverse cell identities and coordinating chromatin organization by profiling transcription factor (TF) combinatorics, identifying TFs with non-cell-autonomous activity, and uncovering TFs underlying higher-order chromatin interactions. Cell-type-specific CREs were enriched for enhancer activity and within unmethylated long terminal repeat retrotransposons. Moreover, we found cell-type-specific CREs are hotspots for phenotype-associated genetic variants and were targeted by selection during modern maize breeding, highlighting the biological implications of this CRE atlas. Through comparison of maize and Arabidopsis thaliana developmental trajectories, we identified TFs and CREs with conserved and divergent chromatin dynamics, showcasing extensive evolution of gene regulatory networks. In addition to this rich dataset, we developed single-cell analysis software, Socrates, which can be used to understand cis-regulatory variation in any species.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Elementos Reguladores de Transcrição/genética , Zea mays/genética , Arabidopsis/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/fisiologia , Redes Reguladoras de Genes/genética , Genoma , Genômica , Elementos Reguladores de Transcrição/fisiologia , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.
Assuntos
Córtex Cerebral/embriologia , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Análise de Célula Única , Astrócitos/citologia , Diferenciação Celular , Linhagem da Célula/genética , Análise por Conglomerados , Aprendizado Profundo , Epigênese Genética , Lógica Fuzzy , Glutamatos/metabolismo , Humanos , Mutação/genética , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
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
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Assuntos
Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Degeneração Neural/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Axônios/metabolismo , Proteína C9orf72/genética , Morte Celular , Células Cultivadas , Córtex Cerebral/patologia , Cromatina/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Drosophila , Camundongos Endogâmicos C57BL , Degeneração Neural/patologia , Estabilidade Proteica , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Córtex Pré-Frontal/embriologia , Telencéfalo/embriologia , Animais , Transtorno Autístico/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Eucromatina/genética , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Ontologia Genética , Predisposição Genética para Doença , Idade Gestacional , Humanos , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , Mutação Puntual , Córtex Pré-Frontal/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise Espaço-Temporal , Telencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.
Assuntos
Condrócitos/fisiologia , Articulação do Joelho/fisiologia , Osteoartrite/genética , Animais , Evolução Biológica , Condrócitos/metabolismo , Evolução Molecular , Predisposição Genética para Doença/genética , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Joelho/fisiologia , Camundongos , Células NIH 3T3 , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de RiscoRESUMO
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
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.
Assuntos
Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Elementos Reguladores de Transcrição/genética , Animais , Sítios de Ligação/genética , Cromatina , Imunoprecipitação da Cromatina/métodos , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.
Assuntos
Receptor Cross-Talk/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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
Non-coding regions comprise most of the human genome and harbor a significant fraction of risk alleles for neuropsychiatric diseases, yet their functions remain poorly defined. We created a high-resolution map of non-coding elements involved in human cortical neurogenesis by contrasting chromatin accessibility and gene expression in the germinal zone and cortical plate of the developing cerebral cortex. We link distal regulatory elements (DREs) to their cognate gene(s) together with chromatin interaction data and show that target genes of human-gained enhancers (HGEs) regulate cortical neurogenesis and are enriched in outer radial glia, a cell type linked to human cortical evolution. We experimentally validate the regulatory effects of predicted enhancers for FGFR2 and EOMES. We observe that common genetic variants associated with educational attainment, risk for neuropsychiatric disease, and intracranial volume are enriched within regulatory elements involved in cortical neurogenesis, demonstrating the importance of this early developmental process for adult human cognitive function.
Assuntos
Córtex Cerebral/metabolismo , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese , Neurônios/metabolismo , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Feminino , Humanos , Masculino , Neurônios/citologia , Polimorfismo Genético , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , RNA , Anêmonas-do-Mar/fisiologia , Actinas/química , Motivos de Aminoácidos , Animais , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Genoma , Genômica , Filogenia , Anêmonas-do-Mar/genética , Análise de Sequência de RNA , Transcriptoma , Tubulina (Proteína)/químicaRESUMO
Neural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This "primary regionalization" allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.
Assuntos
Montagem e Desmontagem da Cromatina , Indução Embrionária , Neurogênese , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
Understanding how HIV-1-infected cells proliferate and persist is key to HIV-1 eradication, but the heterogeneity and rarity of HIV-1-infected cells hamper mechanistic interrogations. Here, we used single-cell DOGMA-seq to simultaneously capture transcription factor accessibility, transcriptome, surface proteins, HIV-1 DNA, and HIV-1 RNA in memory CD4+ T cells from six people living with HIV-1 during viremia and after suppressive antiretroviral therapy. We identified increased transcription factor accessibility in latent HIV-1-infected cells (RORC) and transcriptionally active HIV-1-infected cells (interferon regulatory transcription factor [IRF] and activator protein 1 [AP-1]). A proliferation program (IKZF3, IL21, BIRC5, and MKI67 co-expression) promoted the survival of transcriptionally active HIV-1-infected cells. Both latent and transcriptionally active HIV-1-infected cells had increased IKZF3 (Aiolos) expression. Distinct epigenetic programs drove the heterogeneous cellular states of HIV-1-infected cells: IRF:activation, Eomes:cytotoxic effector differentiation, AP-1:migration, and cell death. Our study revealed the single-cell epigenetic, transcriptional, and protein states of latent and transcriptionally active HIV-1-infected cells and cellular programs promoting HIV-1 persistence.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/genética , HIV-1/fisiologia , Latência Viral/genética , Linfócitos T CD4-Positivos , Fator de Transcrição AP-1 , Epigênese Genética , Fator de Transcrição Ikaros/genéticaRESUMO
CD4+ T cell-derived interleukin 21 (IL-21) sustains CD8+ T cell responses during chronic viral infection, but the helper subset that confers this protection remains unclear. Here, we applied scRNA and ATAC-seq approaches to determine the heterogeneity of IL-21+CD4+ T cells during LCMV clone 13 infection. CD4+ T cells were comprised of three transcriptionally and epigenetically distinct populations: Cxcr6+ Th1 cells, Cxcr5+ Tfh cells, and a previously unrecognized Slamf6+ memory-like (Tml) subset. T cell differentiation was specifically redirected toward the Tml subset during chronic, but not acute, LCMV infection. Although this subset displayed an enhanced capacity to accumulate and some developmental plasticity, it remained largely quiescent, which may hinder its helper potential. Conversely, mixed bone marrow chimera experiments revealed that Tfh cell-derived IL-21 was critical to sustain CD8+ T cell responses and viral control. Thus, strategies that bolster IL-21+Tfh cell responses may prove effective in enhancing CD8+ T cell-mediated immunity.
Assuntos
Células T Auxiliares Foliculares , Viroses , Linfócitos T CD8-Positivos , Humanos , InterleucinasRESUMO
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
Assuntos
Epigenômica , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula , Células Clonais/citologia , Fluorescência , Hematopoese , Inflamação/patologia , Camundongos , Transcrição GênicaRESUMO
Genetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction. However, there were few changes in nascent transcription within the first 6 h of degradation. Nevertheless, we demonstrated a requirement for SMARCA5 to control nucleosome repeat length at G1/S and during the S phase. SMARCA5 co-localized with CTCF and H2A.Z, and we found a rapid loss of CTCF DNA binding and disruption of nucleosomal phasing around CTCF binding sites. This spatiotemporal analysis indicates that SMARCA5 is continuously required for maintaining nucleosomal spacing.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Reparo do DNA , Nucleossomos , Humanos , Adenosina Trifosfatases/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genéticaRESUMO
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.