RESUMEN
Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.
Asunto(s)
Tolerancia Central/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Nucleares/inmunología , Timo/inmunología , Factores de Transcripción/inmunología , Animales , Cromatina , Ratones , Ratones Transgénicos , Proteína AIRERESUMEN
mSWI/SNF or BAF chromatin regulatory complexes are dosage-sensitive regulators of human neural development frequently mutated in autism spectrum disorders and intellectual disability. Cell cycle exit and differentiation of neural stem/progenitor cells is accompanied by BAF subunit switching to generate neuron-specific nBAF complexes. We manipulated the timing of BAF subunit exchange in vivo and found that early loss of the npBAF subunit BAF53a stalls the cell cycle to disrupt neurogenesis. Loss of BAF53a results in decreased chromatin accessibility at specific neural transcription factor binding sites, including the pioneer factors SOX2 and ASCL1, due to Polycomb accumulation. This results in repression of cell cycle genes, thereby blocking cell cycle progression and differentiation. Cell cycle block upon Baf53a deletion could be rescued by premature expression of the nBAF subunit BAF53b but not by other major drivers of proliferation or differentiation. WNT, EGF, bFGF, SOX2, c-MYC, or PAX6 all fail to maintain proliferation in the absence of BAF53a, highlighting a novel mechanism underlying neural progenitor cell cycle exit in the continued presence of extrinsic proliferative cues.
Asunto(s)
Actinas/metabolismo , Ciclo Celular/genética , Corteza Cerebelosa/embriología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Actinas/genética , Animales , Sitios de Unión/genética , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Eliminación de Gen , Genes cdc/genética , Ratones , Neurogénesis/genética , Proteínas del Grupo Polycomb/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.
Asunto(s)
Epigenómica , Heterocromatina/metabolismo , Código de Histonas , Animales , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias , Fibroblastos/metabolismo , Histonas/metabolismo , Cinética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Hipocampo/patología , Actinas/genética , Adenosina Trifosfato/genética , Animales , Trastorno del Espectro Autista/patología , Conducta Animal/fisiología , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Emparejamiento Cromosómico/genética , Emparejamiento Cromosómico/fisiología , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Dendritas/genética , Dendritas/fisiología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Humanos , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/metabolismo , Neuronas/patología , Factores de Transcripción/genéticaRESUMEN
Recent exon-sequencing studies of human tumours have revealed that subunits of BAF (mammalian SWI/SNF) complexes are mutated in more than 20% of all human malignancies, but the mechanisms involved in tumour suppression are unclear. BAF chromatin-remodelling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure and the placement of Polycomb repressive complex 2 (PRC2) across the genome. Several proteins dedicated to this multisubunit complex, including BRG1 (also known as SMARCA4) and BAF250a (also known as ARID1A), are mutated at frequencies similar to those of recognized tumour suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastomas and more than 15% of Burkitt's lymphomas. Here we show a previously unknown function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1 in mouse cells, as well as the expression of BRG1 point mutants identified in human tumours, leads to anaphase bridge formation (in which sister chromatids are linked by catenated strands of DNA) and a G2/M-phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes interact directly with endogenous topoisomerase IIα (TOP2A) through BAF250a and are required for the binding of TOP2A to approximately 12,000 sites across the genome. Our results demonstrate that TOP2A chromatin binding is dependent on the ATPase activity of BRG1, which is compromised in oncogenic BRG1 mutants. These studies indicate that the ability of TOP2A to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumour suppressors.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Encadenado/química , ADN Encadenado/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Anafase , Animales , Antígenos de Neoplasias/genética , Puntos de Control del Ciclo Celular , Cromátides/metabolismo , Ensamble y Desensamble de Cromatina , Segregación Cromosómica , ADN Helicasas/deficiencia , ADN Helicasas/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Fibroblastos , Fase G2 , Células HEK293 , Humanos , Meduloblastoma/genética , Ratones , Mitosis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas de Unión a Poli-ADP-Ribosa , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.
Asunto(s)
ADN Helicasas/genética , Genes Dominantes , Mutación , Proteínas Nucleares/genética , Factores de Transcripción/genética , Adenosina Trifosfatasas/metabolismo , Animales , Cromatina/química , Ensamble y Desensamble de Cromatina , Medios de Cultivo , Elementos de Facilitación Genéticos , Epigenómica , Genotipo , Heterocigoto , Humanos , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Análisis Multivariante , Mutación Missense , Neoplasias/genética , Proteínas del Grupo Polycomb/genética , Análisis de Secuencia de ARNRESUMEN
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
Asunto(s)
Carcinogénesis/genética , Proteínas de Unión al ADN/genética , Heterocromatina/genética , Proteínas Nucleares/genética , Proteínas del Grupo Polycomb/genética , Cromatina/genética , Replicación del ADN/genética , Epigénesis Genética/genética , Humanos , Mutación/genética , ARN Polimerasa II/genética , Transcripción Genética/genéticaRESUMEN
The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN-Topoisomerasas de Tipo II/metabolismo , Heterocromatina/metabolismo , Animales , Inmunoprecipitación de Cromatina , ADN/metabolismo , Electroforesis en Gel de Agar , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Sitios Genéticos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Sirolimus/farmacología , Factores de Tiempo , Sitio de Iniciación de la Transcripción , Transcripción Genética/efectos de los fármacosRESUMEN
Signalling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs). This contrasts with most cell types where STAT3 signalling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent on Brg1, the ATPase subunit of a specialized chromatin remodelling complex (esBAF) found in ESCs. Brg1 is required to establish chromatin accessibility at STAT3 binding targets, preparing these sites to respond to LIF signalling. Brg1 deletion leads to rapid polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg1-activated targets genome wide, including the target genes of the LIF signalling pathway. Hence, one crucial role of Brg1 in ESCs involves its ability to potentiate LIF signalling by opposing PcG. Contrary to expectations, Brg1 also facilitates PcG function at classical PcG targets, including all four Hox loci, reinforcing their repression in ESCs. Therefore, esBAF does not simply antagonize PcG. Rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.