RESUMO
Heterochromatin is characterized by dimethylated or trimethylated histone H3 Lys9 (H3K9me2 or H3K9me3, respectively) and is found at transposable elements, satellite repeats and genes, where it ensures their transcriptional silencing. The histone methyltransferases (HMTs) that methylate H3K9 - in mammals Suppressor of variegation 3-9 homologue 1 (SUV39H1), SUV39H2, SET domain bifurcated 1 (SETDB1), SETDB2, G9A and G9A-like protein (GLP) - and the 'readers' of H3K9me2 or H3K9me3 are highly conserved and show considerable redundancy. Despite their redundancy, genetic ablation or mistargeting of an individual H3K9 methyltransferase can correlate with impaired cell differentiation, loss of tissue identity, premature aging and/or cancer. In this Review, we discuss recent advances in understanding the roles of the known H3K9-specific HMTs in ensuring transcriptional homeostasis during tissue differentiation in mammals. We examine the effects of H3K9-methylation-dependent gene repression in haematopoiesis, muscle differentiation and neurogenesis in mammals, and compare them with mechanistic insights obtained from the study of model organisms, notably Caenorhabditis elegans and Drosophila melanogaster. In all these organisms, H3K9-specific HMTs have both unique and redundant roles that ensure the maintenance of tissue integrity by restricting the binding of transcription factors to lineage-specific promoters and enhancer elements.
Assuntos
Heterocromatina , Histona-Lisina N-Metiltransferase , Animais , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Mamíferos/genética , MetilaçãoRESUMO
Transcriptionally silenced heterochromatin bearing methylation of histone H3 on lysine 9 (H3K9me) is critical for maintaining organismal viability and tissue integrity. Here we show that in addition to ensuring H3K9me, MET-2, the Caenorhabditis elegans homolog of the SETDB1 histone methyltransferase, has a noncatalytic function that contributes to gene repression. Subnuclear foci of MET-2 coincide with H3K9me deposition, yet these foci also form when MET-2 is catalytically deficient and H3K9me is compromised. Whereas met-2 deletion triggers a loss of silencing and increased histone acetylation, foci of catalytically deficient MET-2 maintain silencing of a subset of genes, blocking acetylation on H3K9 and H3K27. In normal development, this noncatalytic MET-2 activity helps to maintain fertility. Under heat stress MET-2 foci disperse, coinciding with increased acetylation and transcriptional derepression. Our study suggests that the noncatalytic, focus-forming function of this SETDB1-like protein and its intrinsically disordered cofactor LIN-61 is physiologically relevant.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Animais Geneticamente Modificados , Biocatálise , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Metilação , Modelos Biológicos , Mutação , Transcrição GênicaRESUMO
The developmental role of histone H3K9 methylation (H3K9me), which typifies heterochromatin, remains unclear. In Caenorhabditis elegans, loss of H3K9me leads to a highly divergent upregulation of genes with tissue and developmental-stage specificity. During development H3K9me is lost from differentiated cell type-specific genes and gained at genes expressed in earlier developmental stages or other tissues. The continuous deposition of H3K9me2 by the SETDB1 homolog MET-2 after terminal differentiation is necessary to maintain repression. In differentiated tissues, H3K9me ensures silencing by restricting the activity of a defined set of transcription factors at promoters and enhancers. Increased chromatin accessibility following the loss of H3K9me is neither sufficient nor necessary to drive transcription. Increased ATAC-seq signal and gene expression correlate at a subset of loci positioned away from the nuclear envelope, while derepressed genes at the nuclear periphery remain poorly accessible despite being transcribed. In conclusion, H3K9me deposition can confer tissue-specific gene expression and maintain the integrity of terminally differentiated muscle by restricting transcription factor activity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sequenciamento de Cromatina por Imunoprecipitação , Perfilação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Metilação , Ligação Proteica , Fatores de Tempo , TranscriptomaRESUMO
The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with the methylation of histone H3 lysine 9 (H3K9me) defining transcriptionally silent heterochromatin. We show here that the C. elegans SET-25 (SUV39/G9a) histone methyltransferase (HMT), which catalyzes H3K9me1, me2 and me3, can establish repressed chromatin domains de novo, unlike the SETDB1 homolog MET-2. Thus, SET-25 is needed to silence novel insertions of RNA or DNA transposons, and repress tissue-specific genes de novo during development. We identify two partially redundant pathways that recruit SET-25 to its targets. One pathway requires LIN-61 (L3MBTL2), which uses its four MBT domains to bind the H3K9me2 deposited by MET-2. The second pathway functions independently of MET-2 and involves the somatic Argonaute NRDE-3 and small RNAs. This pathway targets primarily highly conserved RNA and DNA transposons. These redundant SET-25 targeting pathways (MET-2-LIN-61-SET-25 and NRDE-3-SET-25) ensure repression of intact transposons and de novo insertions, while MET-2 can act alone to repress simple and satellite repeats. Removal of both pathways in the met-2;nrde-3 double mutant leads to the loss of somatic H3K9me2 and me3 and the synergistic derepression of transposons in embryos, strongly elevating embryonic lethality.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica/genética , Heterocromatina/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/genética , Embrião não Mamífero , Inativação Gênica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metilação , Mutação , Proteínas de Ligação a RNA/genéticaRESUMO
In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Exorribonucleases/genética , Histonas/genética , Estabilidade de RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , Caenorhabditis elegans/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Metilação , Proteínas do Grupo Polycomb/genética , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/genética , Deleção de Genes , Distrofia Muscular de Emery-Dreifuss/genética , Animais , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/patologia , Cromatina/genética , Modelos Animais de Doenças , Genoma Helmíntico/genética , Laminina/genética , Laminina/metabolismo , Músculos/fisiopatologia , Distrofia Muscular de Emery-Dreifuss/fisiopatologia , Mutação , Estrutura Terciária de Proteína/genética , Sarcômeros/química , Sarcômeros/genética , Transcrição Gênica/genéticaRESUMO
The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery1. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity2,3. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane4. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me3,5, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.
Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Cromatina/genética , Cromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Animais , Caenorhabditis elegans/anatomia & histologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Eucromatina/genética , Eucromatina/metabolismo , Mutação com Ganho de Função , Genes Reporter/genética , Histona Acetiltransferases/deficiência , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/metabolismo , Intestinos/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Caenorhabditis elegans has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike set-25 mutants, met-2-null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.
Assuntos
Proteína BRCA1/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Animais , Proteína BRCA1/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis/genética , Embrião não Mamífero , Fertilidade/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Repetições de Microssatélites/genética , Mutação , Processamento Pós-Transcricional do RNA/genética , TemperaturaRESUMO
The segregation of the genome into accessible euchromatin and histone H3K9-methylated heterochromatin helps silence repetitive elements and tissue-specific genes. In Caenorhabditis elegans, MET-2, the homologue of mammalian SETDB1, catalyzes H3K9me1 and me2, yet like SETDB1, its regulation is enigmatic. Contrary to the cytosolic enrichment of overexpressed MET-2, we show that endogenous MET-2 is nuclear throughout development, forming perinuclear foci in a cell cycle-dependent manner. Mass spectrometry identified two cofactors that bind MET-2: LIN-65, a highly unstructured protein, and ARLE-14, a conserved GTPase effector. All three factors colocalize in heterochromatic foci. Ablation of lin-65, but not arle-14, mislocalizes and destabilizes MET-2, resulting in decreased H3K9 dimethylation, dispersion of heterochromatic foci, and derepression of MET-2 targets. Mutation of met-2 or lin-65 also disrupts the perinuclear anchoring of genomic heterochromatin. Loss of LIN-65, like that of MET-2, compromises temperature stress resistance and germline integrity, which are both linked to promiscuous repeat transcription and gene expression.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ciclo Celular , Regulação da Expressão Gênica , Resposta ao Choque Térmico , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Transcrição Gênica , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Metilação , Mutação , Ligação ProteicaRESUMO
Histone H3 lysine 9 (H3K9) methylation is a conserved modification that generally represses transcription. In Caenorhabditis elegans it is enriched on silent tissue-specific genes and repetitive elements. In met-2 set-25 double mutants, which lack all H3K9 methylation (H3K9me), embryos differentiate normally, although mutant adults are sterile owing to extensive DNA-damage-driven apoptosis in the germ line. Transposons and simple repeats are derepressed in both germline and somatic tissues. This unprogrammed transcription correlates with increased rates of repeat-specific insertions and deletions, copy number variation, R loops and enhanced sensitivity to replication stress. We propose that H3K9me2 or H3K9me3 stabilizes and protects repeat-rich genomes by suppressing transcription-induced replication stress.
Assuntos
Caenorhabditis elegans/metabolismo , Histonas/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA de Helmintos/metabolismo , Fertilidade , Genes de Helmintos , Histona-Lisina N-Metiltransferase/metabolismo , Sequências Repetitivas Dispersas , Lisina/metabolismo , Metilação , Hibridização de Ácido Nucleico , Estabilidade de RNA , RNA de Helmintos/metabolismo , TemperaturaRESUMO
Eukaryotic genomes contain millions of copies of repetitive elements (RE). Although the euchromatic parts of most genomes are clearly annotated, the repetitive/heterochromatic parts are poorly defined. It is estimated that between 50 and 70% of the human genome is composed of REs. Despite this, we know surprisingly little about the physiological relevance, molecular regulation and the composition of these regions. This primarily reflects the difficulty that REs pose for PCR-based assays, and their poor map-ability in next generation sequencing experiments. Here we first summarize the nature and classification of REs and then examine how this has been used in the recent years to broaden our understanding of mechanisms that keep the repetitive regions of our genomes silent and stable.
Assuntos
Doença/genética , Genoma Humano , Instabilidade Genômica , Sequências Repetitivas de Ácido Nucleico , Animais , Elementos de DNA Transponíveis , Genoma , HumanosRESUMO
Heterochromatin was first defined by Emil Heitz in 1928 by light microscopy. In the 1950s electron microscopy studies revealed that heterochromatin preferentially localizes to the nuclear periphery and around the nucleolus. While the use of genomic approaches led to the genome wide identification of lamina-associated and nucleolus-associated chromatin domains (LADs, NADs), recent studies now shed light on the processes mediating this topology and its dynamics. The identification of different factors on all regulatory levels, such as transcription factors, histone modifications, chromatin proteins, DNA sequences and non-coding RNAs, suggests the involvement of multiple distinct tethering pathways. Positioning at these nuclear sub-compartments is often but not always associated with transcriptional silencing, underlining the importance of the pre-existing chromatin context.
Assuntos
Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Animais , Núcleo Celular/genética , Inativação Gênica , Genoma , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids because their interaction causes lethality in male hybrid offspring. Here, we show that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. We find that the Hmr expression level is substantially higher in Drosophila melanogaster, whereas Lhr expression levels are increased in Drosophila simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalization of the complex, an interference with the regulation of transposable elements, and an impairment of cell proliferation. Our findings provide evidence for a major role of centromere divergence in the generation of biodiversity.
Assuntos
Centrômero/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Fertilidade/genética , Genes Letais , Isolamento Reprodutivo , Animais , Evolução Biológica , Western Blotting , Proliferação de Células , Células Cultivadas , Segregação de Cromossomos , Elementos de DNA Transponíveis/genética , Drosophila/classificação , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus. Centromere clustering was found to depend on centric chromatin rather than specific DNA sequences. Moreover, unclustering of centromeres results in the spatial destabilization of pericentric heterochromatin organization, leading to partial defects in the silencing of repetitive elements, defects during chromosome segregation, and genome instability.
Assuntos
Nucléolo Celular/metabolismo , Centrômero/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nucleoplasminas/metabolismo , Animais , Fator de Ligação a CCCTC , Linhagem Celular , Cromossomos de Insetos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Técnicas de Silenciamento de Genes , Inativação Gênica , Instabilidade Genômica , Hemócitos/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Interfase , Nucleoplasminas/genética , Ligação Proteica , Mapas de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismoRESUMO
CENH3 is a centromere-specific histone H3 variant essential for kinetochore assembly. Despite its central role in centromere function, there has been no conclusive evidence supporting CENH3 as sufficient to determine centromere identity. To address this question, we artificially targeted Drosophila CENH3 (CENP-A/CID) as a CID-GFP-LacI fusion protein to stably integrated lac operator (lacO) arrays. This ectopic CID focus assembles a functional kinetochore and directs incorporation of CID molecules without the LacI-anchor, providing evidence for the self-propagation of the epigenetic mark. CID-GFP-LacI-bound extrachromosomal lacO plasmids can assemble kinetochore proteins and bind microtubules, resulting in their stable transmission for several cell generations even after eliminating CID-GFP-LacI. We conclude that CID is both necessary and sufficient to serve as an epigenetic centromere mark and nucleate heritable centromere function.