RESUMEN
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.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Animales , Humanos , Cromatina , Ensamble y Desensamble de Cromatina , Proteínas Nucleares/metabolismo , Nucleosomas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , RatonesRESUMEN
A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific chromatin assembly that supersedes these constraints is required to configure the genome to guide gene expression changes that drive faithful lineage progression. Loss-of-function approaches in B cell precursors show that IKAROS assembles interactions across megabase distances in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries to assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains. Gain of function in epithelial cells confirms IKAROS' ability to reconfigure chromatin architecture at multiple scales. Although the compaction of the Igκ locus required for genome editing represents a function of IKAROS unique to lymphocytes, the more general function to preconfigure the genome to support lineage-specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction.
Asunto(s)
Cromatina , Genoma , Linfocitos B/metabolismo , Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Humanos , Animales , RatonesRESUMEN
Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.
Asunto(s)
Ensamble y Desensamble de Cromatina , Complejos Multiproteicos , Proteínas Nucleares , Humanos , Cromatina , Proteínas de Unión al ADN/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismoRESUMEN
Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Metilación de ADN , Histonas , Nucleosomas , Ensamble y Desensamble de Cromatina , ADN , Metilasas de Modificación del ADN , Epigénesis Genética , Histonas/genética , Nucleosomas/genética , Semen , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.
Asunto(s)
Cromatina , Nucleosomas , Cromatina/genética , Ensamble y Desensamble de Cromatina , ADN/genética , ADN/metabolismo , Nucleosomas/genética , Factores de Transcripción/genéticaRESUMEN
Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.
Asunto(s)
Cromatina , Placenta , Animales , Factor de Unión a CCCTC/metabolismo , Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Evolución Molecular , Femenino , Genoma , Mamíferos/metabolismo , Placenta/metabolismo , Embarazo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores , Factores de Transcripción , Proteínas Supresoras de Tumor , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ratones Endogámicos C57BL , Proteínas Cromosómicas no Histona/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Noqueados , Ensamble y Desensamble de Cromatina , Diferenciación Celular/inmunologíaRESUMEN
The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism. Whether remodeller activities mainly catalyse nucleosome dynamics or accurately co-determine nucleosome organization remained unclear. In this Review, we discuss the emerging mechanisms of chromatin remodelling: dynamic remodeller architectures and their interactions, the inner workings of the ATPase cycle, allosteric regulation and pathological dysregulation. Recent mechanistic insights argue for a decisive role of remodellers in the energy-driven self-organization of chromatin, which enables both stability and plasticity of genome regulation - for example, during development and stress. Different remodellers, such as members of the SWI/SNF, ISWI, CHD and INO80 families, process (epi)genetic information through specific mechanisms into distinct functional outputs. Combinatorial assembly of remodellers and their interplay with histone modifications, histone variants, DNA sequence or DNA-bound transcription factors regulate nucleosome mobilization or eviction or histone exchange. Such input-output relationships determine specific nucleosome positions and compositions with distinct DNA accessibilities and mediate differential genome regulation. Finally, remodeller genes are often mutated in diseases characterized by genome dysregulation, notably in cancer, and we discuss their physiological relevance.
Asunto(s)
Cromatina , Histonas , Humanos , Histonas/metabolismo , Nucleosomas , Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , ADN , Adenosina Trifosfato/metabolismoRESUMEN
Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Heterocromatina/metabolismo , Proteínas del Grupo Polycomb/genética , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Femenino , Silenciador del Gen , Heterocromatina/química , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermatozoides/citología , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Cromosoma X/químicaRESUMEN
Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4+ T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4+ T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4+ T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue.
Asunto(s)
Envejecimiento , Linfocitos T CD4-Positivos , Factor de Transcripción Ikaros , Anciano , Animales , Humanos , Ratones , Adulto Joven , Envejecimiento/inmunología , Envejecimiento/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Ensamble y Desensamble de Cromatina , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T , Factor de Transcripción STAT5 , Factor de Transcripción Ikaros/metabolismoRESUMEN
The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.
Asunto(s)
Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiología , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Fertilización/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/metabolismo , Oocitos/fisiología , Fosforilación , Protamina Quinasa/genética , Protamina Quinasa/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Empalme del ARN/genética , Empalme del ARN/fisiología , Espermatozoides/metabolismo , Factores de Transcripción/metabolismo , Cigoto/metabolismoRESUMEN
Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.
Asunto(s)
Enfermedad , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Ensamble y Desensamble de Cromatina , Microscopía por Crioelectrón , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enfermedad/genética , Humanos , Mutación Missense/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología Estructural de Proteína , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.
Asunto(s)
Benzamidas/uso terapéutico , Epigénesis Genética/genética , Piridonas/uso terapéutico , Sarcoma/tratamiento farmacológico , Compuestos de Bifenilo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/genética , Inhibidores Enzimáticos/farmacología , Epigenómica , Terapia Genética/métodos , Humanos , Morfolinas , Proteínas Nucleares/metabolismo , Sarcoma/genética , Factores de Transcripción/metabolismoRESUMEN
Regulatory T (Treg) cells require (interleukin-2) IL-2 for their homeostasis by affecting their proliferation, survival and activation. Here we investigated transcriptional and epigenetic changes after acute, periodic and persistent IL-2 receptor (IL-2R) signaling in mouse peripheral Treg cells in vivo using IL-2 or the long-acting IL-2-based biologic mouse IL-2-CD25. We show that initially IL-2R-dependent STAT5 transcription factor-dependent pathways enhanced gene activation, chromatin accessibility and metabolic reprogramming to support Treg cell proliferation. Unexpectedly, at peak proliferation, less accessible chromatin prevailed and was associated with Treg cell contraction. Restimulation of IL-2R signaling after contraction activated signature IL-2-dependent genes and others associated with effector Treg cells, whereas genes associated with signal transduction were downregulated to somewhat temper expansion. Thus, IL-2R-dependent Treg cell homeostasis depends in part on a shift from more accessible chromatin and expansion to less accessible chromatin and contraction. Mouse IL-2-CD25 supported greater expansion and a more extensive transcriptional state than IL-2 in Treg cells, consistent with greater efficacy to control autoimmunity.
Asunto(s)
Ensamble y Desensamble de Cromatina , Interleucina-2 , Linfocitos T Reguladores , Animales , Cromatina/metabolismo , Interleucina-2/metabolismo , Ratones , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transducción de SeñalRESUMEN
A developmental program affecting human face shape is shown by Greenberg et al. (2019) to hinge on the ability to distinguish a single methyl group between two histone variant isoforms and the action of the chromatin-remodeling enzyme SRCAP. This challenges researchers to link atomic structure to a morphological defect.
Asunto(s)
Cromatina , Histonas , Aminoácidos , Ensamble y Desensamble de Cromatina , Humanos , Isoformas de ProteínasRESUMEN
Several general principles of global 3D genome organization have recently been established, including non-random positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, we systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intra-chromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. Our observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Cromatina/genética , Componentes Genómicos/fisiología , Línea Celular , Núcleo Celular/genética , Cromosomas , Fibroblastos/fisiología , Genoma/genética , Componentes Genómicos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Análisis de la Célula IndividualRESUMEN
Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.
Asunto(s)
Blastocisto/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Animales , Femenino , RatonesRESUMEN
Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and ß-rhombus analogous to α helix and ß sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.
Asunto(s)
Cromatina/ultraestructura , Nucleosomas/ultraestructura , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Cromosomas/metabolismo , Cromosomas/ultraestructura , Nucleosomas/genética , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitio de Iniciación de la TranscripciónRESUMEN
The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.
Asunto(s)
Anomalías Múltiples/genética , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Anomalías Craneofaciales/genética , Trastornos del Crecimiento/genética , Defectos del Tabique Interventricular/genética , Histonas/genética , Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Animales , Células Madre Embrionarias , Células HEK293 , Humanos , Mutación , Xenopus laevisRESUMEN
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.