RESUMO
Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.
Assuntos
Linfócitos B/imunologia , Imunidade Humoral/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Complexo Repressor Polycomb 1/imunologia , Proteínas Proto-Oncogênicas/imunologia , Imunidade Adaptativa/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Feminino , Centro Germinativo/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Precise control of gene expression is fundamental to cell function and development. Although ultimately gene expression relies on DNA-binding transcription factors to guide the activity of the transcription machinery to genes, it has also become clear that chromatin and histone post-translational modification have fundamental roles in gene regulation. Polycomb repressive complexes represent a paradigm of chromatin-based gene regulation in animals. The Polycomb repressive system comprises two central protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2, which are essential for normal gene regulation and development. Our early understanding of Polycomb function relied on studies in simple model organisms, but more recently it has become apparent that this system has expanded and diverged in mammals. Detailed studies are now uncovering the molecular mechanisms that enable mammalian PRC1 and PRC2 to identify their target sites in the genome, communicate through feedback mechanisms to create Polycomb chromatin domains and control transcription to regulate gene expression. In this Review, we discuss and contextualize the emerging principles that define how this fascinating chromatin-based system regulates gene expression in mammals.
Assuntos
Regulação da Expressão Gênica/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Metilação , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 2/química , Processamento de Proteína Pós-Traducional , Transcrição Gênica , UbiquitinaçãoRESUMO
The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.
Assuntos
Apoptose , Cromatina/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Azepinas/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Inversão Cromossômica/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , Modelos Animais de Doenças , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.
Assuntos
Cromatina , Complexo Repressor Polycomb 1 , Animais , Camundongos , Cromatina/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Nucleossomos/genética , Ubiquitinação , Expressão Gênica , Mamíferos/metabolismoRESUMO
Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Nucleossomos , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Cromatina/metabolismo , Cromatina/genética , Humanos , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Imagem Individual de MoléculaRESUMO
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
Assuntos
Carcinogênese , Proteínas de Drosophila , Proteólise , Ubiquitina-Proteína Ligases , Animais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Carcinogênese/genética , Humanos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriologia , Genes Supressores de Tumor , Ubiquitinação , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genéticaRESUMO
Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.
Assuntos
Histonas , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Cromatina/genéticaRESUMO
Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase-separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation in living animals. Here, we show in vivo evidence of a critical nonenzymatic repressive function of cPRC1 component CBX2 in the male germline. CBX2 is up-regulated as spermatogonial stem cells differentiate and is required to repress genes that were active in stem cells. CBX2 forms condensates (similar to previously described Polycomb bodies) that colocalize with target genes bound by CBX2 in differentiating spermatogonia. Single-cell analyses of mosaic Cbx2 mutant testes show that CBX2 is specifically required to produce differentiating A1 spermatogonia. Furthermore, the region of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical, which distinguishes this from a mechanism that is reliant on histone modification.
Assuntos
Núcleo Celular , Cromatina , Animais , Masculino , Cromatina/metabolismo , Núcleo Celular/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Espermatogênese/genéticaRESUMO
Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.
Assuntos
DNA/metabolismo , Interferon Tipo I/farmacologia , Lisina/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Fator de Transcrição STAT1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Lisina/genética , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexo Repressor Polycomb 1/genética , Ligação Proteica/efeitos dos fármacos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Estomatite Vesicular/genética , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here, we show that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.
Assuntos
Proteínas de Drosophila , Nucleossomos , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Camundongos , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , RNA/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
Bmi1 is essential for normal and leukemic hematopoiesis, but its target genes in hematopoietic stem cells (HSCs) are incompletely understood. In this issue of Genes & Development, Burgess et al. (pp. 887-900) demonstrate a novel role of Bmi1 in regulating ribosome biogenesis and protein synthesis. Bmi1-deficient HSCs exhibited reduced transplantability, with the up-regulation of ARX and genes involved in ribosome biogenesis. However, depletion of ARX or its known targets, p16 Ink4a /p19 Arf , only partially rescues Bmi1 loss-induced hematopoietic defects. They further demonstrate an increased protein synthesis rate and resultant proteostatic stress in Bmi1 -/- HSCs, indicating a novel mechanism by which Bmi1 controls HSC maintenance.
Assuntos
Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The polycomb complex component Bmi1 promotes the maintenance of stem cells in multiple postnatal tissues, partly by negatively regulating the expression of p16Ink4a and p19Arf, tumor suppressors associated with cellular senescence. However, deficiency for p16Ink4a and p19Arf only partially rescues the function of Bmi1-deficient stem cells. We conditionally deleted Bmi1 from adult hematopoietic cells and found that this slowly depleted hematopoietic stem cells (HSCs). Rather than inducing senescence, Bmi1 deficiency increased HSC division. The increased cell division was caused partly by increased Aristaless-related homeobox (ARX) transcription factor expression, which also increased ribosomal RNA expression. However, ARX deficiency did not rescue HSC depletion. Bmi1 deficiency also increased protein synthesis, protein aggregation, and protein ubiquitylation independent of its effects on cell division and p16Ink4a, p19Arf, and ARX expression. Bmi1 thus promotes HSC quiescence by negatively regulating ARX expression and promotes proteostasis by suppressing protein synthesis. This highlights a new connection between the regulation of stem cell maintenance and proteostasis.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Proteostase , Inibidor p16 de Quinase Dependente de Ciclina/genética , Células-Tronco Hematopoéticas , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Agregados Proteicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Ribossômico/metabolismoRESUMO
The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Assuntos
Cromatina , Proteínas de Drosophila , Animais , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Histonas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Nucleossomos , Drosophila/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismoRESUMO
The Polycomb system modulates chromatin structure to maintain gene repression during cell differentiation. Polycomb repression involves methylation of histone H3K27 (H3K27me3) by Polycomb repressive complex 2 (PRC2), monoubiquitylation of H2A (H2Aub1) by noncanonical PRC1 (ncPRC1), and chromatin compaction by canonical PRC1 (cPRC1), which is independent of its enzymatic activity. Puzzlingly, Polycomb repression also requires deubiquitylation of H2Aub1 by Polycomb repressive deubiquitinase (PR-DUB). In this issue of Genes & Development, Bonnet and colleagues (pp. 1046-1061) resolve this paradox by showing that high levels of H2Aub1 in Drosophila lacking PR-DUB activity promotes open chromatin and gene expression in spite of normal H3K27me3 levels and PRC binding. Pertinently, gene repression is restored by concomitant loss of PRC1 E3 ubiquitin ligase activity but depends on its chromatin compaction activity. These findings suggest that PR-DUB ensures just-right levels of H2Aub1 to allow chromatin compaction by cPRC1.
Assuntos
Proteínas de Drosophila , Histonas , Animais , Proteínas do Grupo Polycomb/genética , Proteínas de Drosophila/genética , Drosophila/genética , Complexo Repressor Polycomb 1/genética , CromatinaRESUMO
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas F-Box/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Desenvolvimento Ósseo , Ilhas de CpG , Proteínas F-Box/química , Proteínas F-Box/genética , Genes Letais , Estudo de Associação Genômica Ampla , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Estrutura Terciária de ProteínaRESUMO
Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.
Assuntos
Enzimas Desubiquitinantes , Histonas , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Humanos , Microscopia Crioeletrônica , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/ultraestrutura , Proteínas do Grupo Polycomb/química , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/ultraestrutura , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/ultraestrutura , Ubiquitinação , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Domínio Catalítico , Enzimas Desubiquitinantes/classificação , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/ultraestrutura , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestruturaRESUMO
BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.
Assuntos
Cromatina/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular/metabolismo , Cromatina/genética , Cromatina/fisiologia , Células-Tronco Embrionárias/metabolismo , Heterocromatina , Histonas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologia , UbiquitinaçãoRESUMO
The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.
Assuntos
Cromatina/química , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Drosophila/metabolismo , Corpos Embrioides , Células-Tronco Embrionárias/citologia , Epigênese Genética , Perfilação da Expressão Gênica , Genômica , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Microscopia Eletrônica , Neurônios/metabolismo , Peptídeos/química , Fenótipo , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 1/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Células-Tronco/citologiaRESUMO
Polycomb repressive complex 1 (PRC1) and PRC2 are critical epigenetic developmental regulators. PRC1 and PRC2 largely overlap in their genomic binding and cooperate to establish repressive chromatin domains demarcated by H2AK119ub and H3K27me3. However, the functional contribution of each complex to gene repression has been a subject of debate, and understanding of its physiological significance requires further studies. Here, using the developing murine epidermis as a paradigm, we uncovered a previously unappreciated functional redundancy between Polycomb complexes. Coablation of PRC1 and PRC2 in embryonic epidermal progenitors resulted in severe defects in epidermal stratification, a phenotype not observed in the single PRC1-null or PRC2-null epidermis. Molecular dissection indicated a loss of epidermal identity that was coupled to a strong derepression of nonlineage transcription factors, otherwise repressed by either PRC1 or PRC2 in the absence of its counterpart. Ectopic expression of subsets of PRC1/2-repressed nonepidermal transcription factors in wild-type epidermal stem cells was sufficient to suppress epidermal identity genes, highlighting the importance of functional redundancy between PRC1 and PRC2. Altogether, our studies show how PRC1 and PRC2 function as two independent counterparts, thereby providing a repressive safety net that protects and preserves lineage identity.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células Epidérmicas/citologia , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4-6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.