RESUMO
Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.
Assuntos
Linfócitos T CD8-Positivos , Proteínas de Ligação a DNA , Interferon Tipo I , Proteínas de Membrana , Neoplasias , Transdução de Sinais , Fatores de Transcrição , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Mutação , Neoplasias/imunologia , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Masculino , Quimiocinas/genética , Quimiocinas/metabolismoRESUMO
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
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.
Assuntos
Montagem e Desmontagem da Cromatina , Complexos Multiproteicos , Proteínas Nucleares , Humanos , Cromatina , Proteínas de Ligação a DNA/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
Assuntos
Infecções por Coronavirus/genética , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Coronavirus/classificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Vero , Internalização do VírusRESUMO
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.
Assuntos
Doença , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Doença/genética , Humanos , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , 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
Macrophages elicit immune responses to pathogens through induction of inflammatory genes. Here, we examined the role of three variants of the SWI/SNF nucleosome remodeling complex-cBAF, ncBAF, and PBAF-in the macrophage response to bacterial endotoxin (lipid A). All three SWI/SNF variants were prebound in macrophages and retargeted to genomic sites undergoing changes in chromatin accessibility following stimulation. Cooperative binding of all three variants associated with de novo chromatin opening and latent enhancer activation. Isolated binding of ncBAF and PBAF, in contrast, associated with activation and repression of active enhancers, respectively. Chemical and genetic perturbations of variant-specific subunits revealed pathway-specific regulation in the activation of lipid A response genes, corresponding to requirement for cBAF and ncBAF in inflammatory and interferon-stimulated gene (ISG) activation, respectively, consistent with differential engagement of SWI/SNF variants by signal-responsive transcription factors. Thus, functional diversity among SWI/SNF variants enables increased regulatory control of innate immune transcriptional programs, with potential for specific therapeutic targeting.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Elementos Facilitadores Genéticos , Inflamação , Macrófagos , Fatores de Transcrição , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Inflamação/imunologia , Inflamação/genética , Elementos Facilitadores Genéticos/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Imunidade Inata , HumanosRESUMO
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/química , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Drosophila/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Espectrometria de Massas , Mutagênese , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CD8+ T cell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused in vivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in Tex-cell differentiation. Depletion of the canonical SWI/SNF form, BAF, impaired initial CD8+ T cell responses in acute and chronic infection. In contrast, disruption of PBAF enhanced Tex-cell proliferation and survival. Mechanistically, PBAF regulated the epigenetic and transcriptional transition from TCF-1+ progenitor Tex cells to more differentiated TCF-1- Tex subsets. Whereas PBAF acted to preserve Tex progenitor biology, BAF was required to generate effector-like Tex cells, suggesting that the balance of these factors coordinates Tex-cell subset differentiation. Targeting PBAF improved tumor control both alone and in combination with anti-PD-L1 immunotherapy. Thus, PBAF may present a therapeutic target in cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Montagem e Desmontagem da Cromatina , Cromatina , Diferenciação Celular , Epigênese GenéticaRESUMO
Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Nucleossomos , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Transcrição Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nucleossomos/metabolismo , Nucleossomos/genética , Regulação Fúngica da Expressão Gênica , Sítio de Iniciação de Transcrição , Cromatina/metabolismo , Cromatina/genéticaRESUMO
Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanisms remain unclear. Here, we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of histone deacetylase (HDAC) activity or the direct targeting of a histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects. The requirement for HDAC in heterochromatin silencing can be bypassed by impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance, providing insights into genome regulatory mechanisms relevant to human diseases.
RESUMO
RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.
Assuntos
Linhagem da Célula , DNA Helicases , Elementos Facilitadores Genéticos , Proteínas Nucleares , Fatores de Transcrição , Animais , Humanos , Linhagem da Célula/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The discovery of epigenetic modulators (writers, erasers, readers, and remodelers) has shed light on previously underappreciated biological mechanisms that promote diseases. With these insights, novel biomarkers and innovative combination therapies can be used to address challenging and difficult to treat disease states. This review highlights key mechanisms that epigenetic writers, erasers, readers, and remodelers control, as well as their connection with disease states and recent advances in associated epigenetic therapies.
Assuntos
Epigênese Genética , Humanos , Animais , Metilação de DNA/genética , Doença/genéticaRESUMO
Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/genética , Ativação TranscricionalRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina , Neoplasias , Animais , Humanos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/genética , Mamíferos/metabolismoRESUMO
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas F-Box , Histona-Lisina N-Metiltransferase , Histona Desmetilases com o Domínio Jumonji , Proteínas do Grupo Polycomb , Proteína SMARCB1 , Cromatina/genética , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Células Tumorais Cultivadas/metabolismoRESUMO
Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, we demonstrate that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, our results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity.
Assuntos
Cromatina , Nucleossomos , Animais , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Zeladoria , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
Polycomb group (PcG) proteins are essential for post-implantation development by depositing repressive histone modifications at promoters, mainly CpG islands (CGIs), of developmental regulator genes. However, promoter PcG marks are erased after fertilization and de novo established in peri-implantation embryos, coinciding with the transition from naive to primed pluripotency. Nevertheless, the molecular basis for this establishment remains unknown. In this study, we show that the expression of the long KDM2B isoform (KDM2BLF), which contains the demethylase domain, is specifically induced at peri-implantation and that its H3K36me2 demethylase activity is required for PcG enrichment at CGIs. Moreover, KDM2BLF interacts with BRG1/BRM-associated factor (BAF) and stabilizes BAF occupancy at CGIs for subsequent gain of accessibility, which precedes PcG enrichment. Consistently, KDM2BLF inactivation results in significantly delayed post-implantation development. In summary, our data unveil dynamic chromatin configuration of CGIs during exit from naive pluripotency and provide a conceptual framework for the spatiotemporal establishment of PcG functions.
Assuntos
Cromatina , Proteínas de Drosophila , Ilhas de CpG , Proteínas de Drosophila/metabolismo , Código das Histonas , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras GenéticasRESUMO
A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).