RESUMO
Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.
Assuntos
Diferenciação Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Camundongos Knockout , Modelos Genéticos , Mutação , Complexo Repressor Polycomb 2/genética , Interferência de RNARESUMO
G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.
Assuntos
Inativação Gênica , Antígenos de Histocompatibilidade/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica , Células HeLa , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Regiões Promotoras GenéticasRESUMO
Long non-coding RNAs (lncRNAs) play diverse roles in physiological and pathological processes. Several lncRNAs have been suggested to modulate gene expression by guiding chromatin-modifying complexes to specific sites in the genome. However, besides the example of Xist, clear-cut evidence demonstrating this novel mode of regulation remains sparse. Here, we focus on HOTAIR, a lncRNA that is overexpressed in several tumor types and previously proposed to play a key role in gene silencing through direct recruitment of Polycomb Repressive Complex 2 (PRC2) to defined genomic loci. Using genetic tools and a novel RNA-tethering system, we investigated the interplay between HOTAIR and PRC2 in gene silencing. Surprisingly, we observed that forced overexpression of HOTAIR in breast cancer cells leads to subtle transcriptomic changes that appear to be independent of PRC2. Mechanistically, we found that artificial tethering of HOTAIR to chromatin causes transcriptional repression, but that this effect does not require PRC2. Instead, PRC2 recruitment appears to be a consequence of gene silencing. We propose that PRC2 binding to RNA might serve functions other than chromatin targeting.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Transcrição Gênica , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genéticaRESUMO
Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.
Assuntos
Cromatina/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Animais , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Complexo Repressor Polycomb 1 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Transdiferenciação Celular , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Neurônios Motores/citologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Desdiferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sistema Digestório/citologia , Histona Desmetilases/química , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Metilação , Modelos Biológicos , Dados de Sequência Molecular , Fatores de Transcrição/metabolismoRESUMO
Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromere transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.
Assuntos
Centrômero/genética , Interferência de RNA , Splicing de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Genes Fúngicos , Heterocromatina/metabolismo , Histonas/metabolismo , Metilação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Spliceossomos/metabolismoRESUMO
Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
Assuntos
Cromatina/metabolismo , Interferência de RNA/fisiologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Centrômero/genética , Centrômero/metabolismo , Cromatina/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transcrição Gênica/fisiologiaRESUMO
Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2.