RESUMO
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
Polycomb group proteins (PcGs) maintain transcriptional repression to preserve cellular identity in two distinct repressive complexes, PRC1 and PRC2, that modify histones by depositing H2AK119ub1 and H3K27me3, respectively. PRC1 and PRC2 exist in different variants and show a complex regulatory cross-talk. However, the contribution that H2AK119ub1 plays in mediating PcG repressive functions remains largely controversial. Using a fully catalytic inactive RING1B mutant, we demonstrated that H2AK119ub1 deposition is essential to maintain PcG-target gene repression in embryonic stem cells (ESCs). Loss of H2AK119ub1 induced a rapid displacement of PRC2 activity and a loss of H3K27me3 deposition. This preferentially affected PRC2.2 variant with respect to PRC2.1, destabilizing canonical PRC1 activity. Finally, we found that variant PRC1 forms can sense H2AK119ub1 deposition, which contributes to their stabilization specifically at sites where this modification is highly enriched. Overall, our data place H2AK119ub1 deposition as a central hub that mounts PcG repressive machineries to preserve cell transcriptional identity.
Assuntos
Regulação da Expressão Gênica , Histonas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Transcrição Gênica , Ubiquitinação , Linhagem Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Mutação de Sentido Incorreto , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.
Assuntos
Complexo Repressor Polycomb 1/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cromatina/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição E2F6/genética , Heterocromatina/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Proteínas Repressoras/genética , Fator de Transcrição DP1/genética , Fatores de Transcrição/genética , Fatores Estimuladores Upstream/genéticaRESUMO
BACKGROUND & AIMS: WNT signaling is central to spatial tissue arrangement and regulating stem cell activity, and it represents the hallmark of gastrointestinal cancers. Although its role in driving intestinal tumors is well characterized, WNT's role in gastric tumorigenesis remains elusive. METHODS: We have developed mouse models to control the specific expression of an oncogenic form of ß-catenin (CTNNB1) in combination with MYC activation in Lgr5+ cells of the gastric antrum. We used multiomics approaches applied in vivo and in organoid models to characterize their cooperation in driving gastric tumorigenesis. RESULTS: We report that constitutive ß-catenin stabilization in the stomach has negligible oncogenic effects and requires MYC activation to induce gastric tumor formation. Although physiologically low MYC levels in gastric glands limit ß-catenin transcriptional activity, increased MYC expression unleashes the WNT oncogenic transcriptional program, promoting ß-catenin enhancer invasion without a direct transcriptional cooperation. MYC activation induces a metabolic rewiring that suppresses lysosomal biogenesis through mTOR and ERK activation and MiT/TFE inhibition. This prevents EPCAM degradation by macropinocytosis, promoting ß-catenin chromatin accumulation and activation of WNT oncogenic transcription. CONCLUSION: Our results uncovered a new signaling framework with important implications for the control of gastric epithelial architecture and WNT-dependent oncogenic transformation.
RESUMO
The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
Assuntos
Enterócitos/metabolismo , Histonas/metabolismo , Intestino Delgado/citologia , Celulas de Paneth/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Animais , Catepsina L/metabolismo , Diferenciação Celular , Homeostase , Mucosa Intestinal/citologia , Camundongos , Microvilosidades/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Organoides , Domínios Proteicos , Tripsina/metabolismoRESUMO
In mammals, commitment and specification of germ cell lines involves complex programs that include sex differentiation, control of proliferation, and meiotic initiation. Regulation of these processes is genetically controlled by fine-tuned mechanisms of gene regulation in which microRNAs (miRNAs) are involved. We have characterized, by small-RNA-seq and bioinformatics analyses, the miRNA expression patterns of male and female mouse primordial germ cells (PGCs) and gonadal somatic cells at embryonic stages E11.5, E12.5, and E13.5. Differential expression analyses revealed differences in the regulation of key miRNA clusters such as miR-199-214, miR-182-183-96, and miR-34c-5p, whose targets have defined roles during gonadal sexual determination in both germ and somatic cells. Extensive analyses of miRNA sequences revealed an increase in noncanonical isoforms on PGCs at E12.5 and dramatic changes of 3' isomiR expression and 3' nontemplate nucleotide additions in female PGCs at E13.5. Additionally, RT-qPCR analyses of genes encoding proteins involved in miRNA biogenesis and 3' nucleotide addition uncovered sexually and developmentally specific expression, characterized by the decay of Drosha, Dgcr8, and Xpo5 expression along gonadal development. These results demonstrate that miRNAs, their isomiRs, and miRNA machinery are differentially regulated and participate actively in gonadal sexual differentiation in both PGCs and gonadal somatic cells.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Diferenciação Sexual/genética , Animais , Diferenciação Celular/genética , Biologia Computacional , Feminino , Células Germinativas , Gônadas/crescimento & desenvolvimento , Carioferinas , Masculino , Camundongos , Ovário/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Ribonuclease III , Testículo/crescimento & desenvolvimentoRESUMO
piRNAs are small non-coding RNAs known to play a main role in defence against transposable elements in germ cells. However, other potential functions, such as biogenesis and differences in somatic and germline expression of these regulatory elements, are not yet fully unravelled. Here, we analysed a variety of piRNA sequences detected in mouse male and female primordial germ cells (PGCs) and gonadal somatic cells at crucial stages during embryonic differentiation of germ cells (11.5-13.5 days post-coitum). NGS of sncRNA and bioinformatic characterization of piRNAs from PGCs and somatic cells, in addition to piRNAs associated with TEs, indicated functional diversification in both cell types. Differences in the proportion of the diverse types of piRNAs are detected between somatic and germline during development. However, the global diversified patterns of piRNA expression are mainly shared between germ and somatic cells, we identified piRNAs related with molecules involved in ribosome components and translation pathway, including piRNAs derived from rRNA (34%), tRNA (10%) and snoRNA (8%). piRNAs from both tRNA and snoRNA are mainly derived from 3' and 5' end regions. These connections between piRNAs and rRNAs, tRNAs or snoRNAs suggest important functions of specialized piRNAs in translation regulation during this window of gonadal development.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Gônadas/embriologia , Gônadas/metabolismo , RNA Interferente Pequeno/genética , Animais , Mapeamento Cromossômico , Biologia Computacional/métodos , Elementos de DNA Transponíveis , Feminino , Genes de RNAr , Genômica/métodos , Células Germinativas/citologia , Masculino , Camundongos , Interferência de RNA , RNA de Transferência/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
Cell fate is determined by specific transcription programs that are essential for tissue homeostasis and regeneration. The E3-ligases RING1A and B represent the core activity of the Polycomb repressive complex 1 (PRC1) that deposits repressive histone H2AK119 mono-ubiquitination (H2AK119ub1), which is essential for mouse intestinal homeostasis by preserving stem cell functions. However, the specific role of different PRC1 forms, which are defined by the six distinct PCGF1-6 paralogs, remains largely unexplored in vivo. We report that PCGF6 regulates mouse intestinal Tuft cell differentiation independently of H2AK119ub1 deposition. We show that PCGF6 chromatin occupancy expands outside Polycomb repressive domains, associating with unique promoter and distal regulatory elements. This occurs in the absence of RING1A/B and involves MGA-mediated E-BOX recognition and specific H3K9me2 promoter deposition. PCGF6 inactivation induces an epithelial autonomous accumulation of Tuft cells that was not phenocopied by RING1A/B loss. This involves direct PCGF6 association with a Tuft cell differentiation program that identified Polycomb-independent properties of PCGF6 in adult tissues homeostasis.
Assuntos
Complexo Repressor Polycomb 1 , Células em Tufo , Animais , Camundongos , Diferenciação Celular/fisiologia , Proteínas do Grupo Polycomb , Ubiquitina-Proteína LigasesRESUMO
Although mitochondria are widely studied organelles, the recent interest in the role of mitochondrial small noncoding RNAs (sncRNAs), miRNAs, and more recently, piRNAs, is providing new functional perspectives in germ cell development and differentiation. piRNAs (PIWI-interacting RNAs) are single-stranded sncRNAs of mostly about 20-35 nucleotides, generated from the processing of pre-piRNAs. We leverage next-generation sequencing data obtained from mouse primordial germ cells and somatic cells purified from early-differentiating embryonic ovaries and testis from 11.5 to 13.5 days postcoitum. Using bioinformatic tools, we elucidate (i) the origins of piRNAs as transcribed from mitochondrial DNA fragments inserted in the nucleus or from the mitochondrial genome; (ii) their levels of expression; and (iii) their potential roles, as well as their association with genomic regions encoding other sncRNAs (such as tRNAs and rRNAs) and the mitochondrial regulatory region (D-loop). Finally, our results suggest how nucleo-mitochondrial communication, both anterograde and retrograde signaling, may be mediated by mitochondria-associated piRNAs.
Assuntos
Pequeno RNA não Traduzido , Testículo , Animais , Células Germinativas/metabolismo , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , Testículo/metabolismoRESUMO
Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/ß-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel diseaserelated mental symptoms may thus be the consequence of a deregulated gutbrain vascular axis.
Assuntos
Plexo Corióideo/irrigação sanguínea , Plexo Corióideo/fisiopatologia , Colite Ulcerativa/fisiopatologia , Colite Ulcerativa/psicologia , Intestinos/fisiopatologia , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Animais , Ansiedade/etiologia , Ansiedade/fisiopatologia , Barreira Hematoencefálica/patologia , Colite Ulcerativa/complicações , Dextranos , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos , Transtornos da Memória/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Transdução de Sinais , Junções Íntimas/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Polycomb repressive complexes are evolutionarily conserved complexes that maintain transcriptional repression during development and differentiation to establish and preserve cell identity. We recently described the fundamental role of PRC1 in preserving intestinal stem cell identity through the inhibition of non-lineage-specific transcription factors. To further elucidate the role of PRC1 in adult stem cell maintenance, we now investigated its role in LGR5+ hair follicle stem cells during regeneration. We show that PRC1 depletion severely affects hair regeneration and, different from intestinal stem cells, derepression of its targets induces the ectopic activation of an epidermal-specific program. Our data support a general role of PRC1 in preserving stem cell identity that is shared between different compartments. However, the final outcome of the ectopic activation of non-lineage-specific transcription factors observed upon loss of PRC1 is largely context-dependent and likely related to the transcription factors repertoire and specific epigenetic landscape of different cellular compartments.
Assuntos
Folículo Piloso/citologia , Intestinos/citologia , Complexo Repressor Polycomb 1/metabolismo , Células-Tronco/citologia , Transcrição Gênica , Animais , Linhagem da Célula , Separação Celular , Cruzamentos Genéticos , Progressão da Doença , Epiderme/metabolismo , Feminino , Citometria de Fluxo , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Ligação Proteica , RNA-Seq , Regeneração , Transdução de SinaisRESUMO
The microenvironment influences cancer drug response and sustains resistance to therapies targeting receptor-tyrosine kinases. However, if and how the tumor microenvironment can be altered during treatment, contributing to resistance onset, is not known. We show that, under prolonged treatment with tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a metabolic shift toward increased glycolysis and lactate production. We identified secreted lactate as the key molecule instructing cancer-associated fibroblasts to produce hepatocyte growth factor (HGF) in a nuclear factor κB-dependent manner. Increased HGF, activating MET-dependent signaling in cancer cells, sustained resistance to TKIs. Functional or pharmacological targeting of molecules involved in the lactate axis abrogated in vivo resistance, demonstrating the crucial role of this metabolite in the adaptive process. This adaptive resistance mechanism was observed in lung cancer patients progressed on EGFR TKIs, demonstrating the clinical relevance of our findings and opening novel scenarios in the challenge to drug resistance.