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1.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
2.
Nat Commun ; 12(1): 4359, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272378

RESUMO

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.


Assuntos
Fibroblastos/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Sistemas CRISPR-Cas , Sequenciamento de Cromatina por Imunoprecipitação , Cromatografia Líquida , Desmetilação , Epigênese Genética , Fibroblastos/enzimologia , Deleção de Genes , Heterocromatina/enzimologia , Heterocromatina/genética , Heterocromatina/ultraestrutura , Histona-Lisina N-Metiltransferase/genética , Hibridização in Situ Fluorescente , Espectrometria de Massas , Metilação , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Processamento de Proteína Pós-Traducional/genética , RNA-Seq , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Transdução de Sinais/genética
3.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203193

RESUMO

Chromatin consists of a complex of DNA and histone proteins as its core components and plays an important role in both packaging DNA and regulating DNA metabolic pathways such as DNA replication, transcription, recombination, and chromosome segregation. Proper functioning of chromatin further involves a network of interactions among molecular complexes that modify chromatin structure and organization to affect the accessibility of DNA to transcription factors leading to the activation or repression of the transcription of target DNA loci. Based on its structure and compaction state, chromatin is categorized into euchromatin, heterochromatin, and centromeric chromatin. In this review, we discuss distinct chromatin factors and molecular complexes that constitute euchromatin-open chromatin structure associated with active transcription; heterochromatin-less accessible chromatin associated with silencing; centromeric chromatin-the site of spindle binding in chromosome segregation.


Assuntos
Centrômero/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Animais , Centrômero/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Eucromatina/genética , Heterocromatina/genética , Humanos
4.
Nucleic Acids Res ; 49(13): 7406-7423, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214177

RESUMO

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Heterocromatina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Caseína Quinase II/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cricetinae , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Fosforilação , Serina/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/fisiologia
5.
Biochim Biophys Acta Gene Regul Mech ; 1864(8): 194725, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34174495

RESUMO

The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin features in situ.


Assuntos
Epigênese Genética , Transferência Ressonante de Energia de Fluorescência , Heterocromatina/metabolismo , Metilação de DNA , Corantes Fluorescentes , Células HEK293 , Código das Histonas , Histonas/metabolismo , Humanos , Análise de Célula Única
6.
PLoS Genet ; 17(6): e1009601, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34086674

RESUMO

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histonas/genética , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Evolução Molecular , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilação , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transgenes
7.
PLoS Genet ; 17(6): e1009645, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34157021

RESUMO

Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Schizosaccharomyces/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Alelos , Substituição de Aminoácidos , Centrômero/química , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Perfilação da Expressão Gênica , Heterocromatina/química , Heterocromatina/metabolismo , Cinética , Modelos Genéticos , Anotação de Sequência Molecular , Mutação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
8.
PLoS Genet ; 17(6): e1009646, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34166371

RESUMO

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Epigênese Genética , Heterocromatina/química , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Centrômero/química , Centrômero/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/deficiência , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Meiose , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Óvulo/citologia , Óvulo/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Nat Commun ; 12(1): 2490, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941775

RESUMO

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Animais , Linhagem Celular , Cristalografia por Raios X , Genoma/genética , Instabilidade Genômica/genética , Heterocromatina/genética , Camundongos
11.
Nat Commun ; 12(1): 2683, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976212

RESUMO

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Heterocromatina/genética , Histonas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Variação Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Sequenciamento Completo do Genoma/métodos
12.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33998651

RESUMO

Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Pareamento Cromossômico/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Espermatócitos/crescimento & desenvolvimento , Espermatogênese/genética , Ubiquitina-Proteína Ligases/genética , Células-Tronco Germinativas Adultas/citologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Epigênese Genética/genética , Heterocromatina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Espermatócitos/fisiologia , Espermatogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
13.
Nature ; 593(7858): 289-293, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33854237

RESUMO

Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown1,2. Here, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, we show that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. Our work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Posicionamento Cromossômico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Genoma de Inseto/genética , Conformação Molecular , Animais , Imunoprecipitação da Cromatina , Cromossomos de Insetos/química , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Hibridização in Situ Fluorescente
14.
Mol Cell ; 81(10): 2216-2230.e10, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33848455

RESUMO

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Sequência de Bases , Reparo do DNA por Junção de Extremidades , Eucromatina/metabolismo , Rearranjo Gênico , Genoma Humano , Heterocromatina/metabolismo , Humanos , Mutação INDEL/genética , Células K562 , Cinética , Ligação Proteica , Reprodutibilidade dos Testes
15.
Plant Cell ; 33(4): 961-979, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-33793815

RESUMO

Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Instabilidade Genômica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Acetilação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Inativação Gênica , Genoma de Planta , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Lisina/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
PLoS Biol ; 19(4): e3001101, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33793547

RESUMO

Here, we reveal that the regulation of Drosophila odorant receptor (OR) expression during the pupal stage is permissive and imprecise. We found that directly after hatching an OR feedback mechanism both directs and refines OR expression. We demonstrate that, as in mice, dLsd1 and Su(var)3-9 balance heterochromatin formation to direct OR expression. We show that the expressed OR induces dLsd1 and Su(var)3-9 expression, linking OR level and possibly function to OR expression. OR expression refinement shows a restricted duration, suggesting that a gene regulatory critical period brings olfactory sensory neuron differentiation to an end. Consistent with a change in differentiation, stress during the critical period represses dLsd1 and Su(var)3-9 expression and makes the early permissive OR expression permanent. This induced permissive gene regulatory state makes OR expression resilient to stress later in life. Hence, during a critical period OR feedback, similar to in mouse OR selection, defines adult OR expression in Drosophila.


Assuntos
Drosophila , Neurogênese/genética , Neurônios Receptores Olfatórios/fisiologia , Receptores Odorantes/fisiologia , Estresse Fisiológico/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Retroalimentação Fisiológica/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Humanos , Masculino , Neurogênese/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Pupa , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo
17.
PLoS Pathog ; 17(4): e1009567, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909709

RESUMO

Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection.


Assuntos
Herpesvirus Humano 1/fisiologia , Replicação Viral/genética , Proteína Nuclear Ligada ao X/fisiologia , Células Cultivadas , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Herpes Simples/genética , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Fenômenos Fisiológicos Virais/genética
18.
Cytogenet Genome Res ; 161(1-2): 52-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33887732

RESUMO

With 82 species currently described, the genus Leptodactylus is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 Leptodactylus species (L. macrosternum, L. pentadactylus, L. fuscus, and Leptodactylus cf. podicipinus), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for Leptodactylus cf. podicipinus which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in L. pentadactylus), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.


Assuntos
Anuros/genética , Cromossomos/ultraestrutura , DNA Ribossômico/genética , Cariotipagem , Repetições de Microssatélites , Animais , Bandeamento Cromossômico , Inversão Cromossômica , Análise Citogenética , Citogenética , Sondas de DNA , Feminino , Geografia , Heterocromatina/metabolismo , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Meiose , Mitose , Região Organizadora do Nucléolo , Filogenia , Especificidade da Espécie
19.
Science ; 372(6540): 371-378, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33888635

RESUMO

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.


Assuntos
Período de Replicação do DNA , Epigênese Genética , Epigenoma , Proteínas de Ligação a Telômeros/metabolismo , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Replicação do DNA , Expressão Gênica , Técnicas de Inativação de Genes , Genoma Humano , Heterocromatina/metabolismo , Código das Histonas , Histonas/metabolismo , Humanos , Proteínas de Ligação a Telômeros/genética
20.
Nucleic Acids Res ; 49(8): 4203-4219, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33706382

RESUMO

Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.


Assuntos
Envelhecimento/metabolismo , Heterocromatina/metabolismo , Sirtuína 3/fisiologia , Envelhecimento/genética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Células Cultivadas , Senescência Celular/genética , Senescência Celular/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Heterocromatina/genética , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Nus , Camundongos SCID , Membrana Nuclear/metabolismo , Domínios Proteicos , Sirtuína 3/química , Sirtuína 3/genética
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