RESUMO
Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Complexo Repressor Polycomb 2/metabolismo , Espermatócitos/citologia , Transcriptoma/genética , Animais , Cromossomos/genética , Cromossomos/metabolismo , Inativação Gênica , Infertilidade Masculina/genética , Laminas/genética , Masculino , Meiose/genética , Camundongos , Complexo Repressor Polycomb 2/genéticaRESUMO
Meiotic silencing of unpaired chromatin (MSUC) occurs during the first meiotic prophase, as chromosomes that fail to pair are sequestered into a transcriptionally repressive nuclear domain. This phenomenon is exemplified by the heterologous sex chromosomes of male mammals, where the ATR DNA damage response kinase is crucial for this silencing event. However, the mechanisms underlying the initiation of MSUC remain unknown. Here, we show that essential components of ATR signaling in murine somatic cells are spatially confined to unpaired chromosomes in spermatocytes, including the ATR-dependent phosphorylation of the single-stranded DNA (ssDNA)-binding complex replication protein A (RPA) and the checkpoint kinase CHK1. These observations support a model in which ssDNA plays a central role in the recruitment of ATR during MSUC, and provide a link to meiotic progression through activation of CHK1.
Assuntos
Pareamento Cromossômico , Cromossomos de Mamíferos/metabolismo , Transdução de Sinais , Espermatócitos/metabolismo , Animais , Especificidade de Anticorpos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Cultivadas , Quinase 1 do Ponto de Checagem , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Masculino , Meiose , Camundongos , Fosforilação , Fosfosserina/metabolismo , Proteínas Quinases/metabolismo , Transporte Proteico , Proteína de Replicação A/metabolismo , Cromossomos Sexuais/metabolismoRESUMO
Germ cell development and gametogenesis require genome-wide transitions in epigenetic modifications and chromatin structure. These changes include covalent modifications to the DNA and histones as well as remodeling activities. Here, we explore the role of the mammalian SWI/SNF chromatin-remodeling complex during spermatogenesis using a conditional allele of the ATPase subunit, brahma-related gene 1 (Brg1, or Smarca4). Not only do BRG1 levels peak during the early stages of meiosis, genetic ablation of Brg1 in murine embryonic gonocytes results in arrest during prophase of meiosis I. Coincident with the timing of meiotic arrest, mutant spermatocytes accumulate unrepaired DNA and fail to complete synapsis. Furthermore, mutant spermatocytes show global alterations to histone modifications and chromatin structure indicative of a more heterochromatic genome. Together, these data demonstrate a requirement for BRG1 activity in spermatogenesis, and suggest a role for the mammalian SWI/SNF complex in programmed recombination and repair events that take place during meiosis.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Espermatogênese , Fatores de Transcrição/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proliferação de Células , Cromatina/fisiologia , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , DNA Helicases/biossíntese , DNA Helicases/genética , Reparo do DNA , Células Germinativas/metabolismo , Histonas/genética , Histonas/metabolismo , Masculino , Meiose/genética , Camundongos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Espermatogênese/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals.
Assuntos
Nucléolo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , DNA Ribossômico/genética , Heterocromatina/genética , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Linhagem Celular , Nucléolo Celular/metabolismo , Dactinomicina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Camundongos , Nucleossomos/genética , Nucleossomos/metabolismo , Pseudogenes/genética , RNA Polimerase I/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transgenes/genéticaRESUMO
The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19DeltaDMD and H19Delta3.8kb-5'H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19DeltaDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5' of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19DeltaDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.
Assuntos
Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Alelos , Animais , Animais Recém-Nascidos , Sequência de Bases , Blastocisto/metabolismo , DNA/genética , Metilação de DNA , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , RNA Longo não Codificante , Deleção de SequênciaRESUMO
The organization of the genome within the mammalian nucleus is nonrandom, with physiologic processes often concentrated in specific three-dimensional domains. This organization may be functionally related to gene regulation and, as such, may play a role in normal development and human disease processes. However, the mechanisms that participate in nuclear organization are poorly understood. Here, we present data characterizing localization of the imprinted Kcnq1 alleles. We show that nucleolar association of the paternal allele (1) is stimulated during the differentiation of trophoblast stem cells, (ii) is dependent upon the Kcnq1ot1 noncoding RNA, (3) does not require polycomb repressive complex 2, and (4) is not sufficient to preclude transcription of imprinted genes. Although nucleolar positioning has been proposed as a mechanism to related to gene silencing, we find that silencing and perinucleolar localization through the Kcnq1ot1 noncoding RNA are separable events.
Assuntos
Nucléolo Celular/metabolismo , Canal de Potássio KCNQ1/genética , Alelos , Animais , Diferenciação Celular , Células Cultivadas , Expressão Gênica/genética , Inativação Gênica , Loci Gênicos , Canal de Potássio KCNQ1/análise , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA não Traduzido/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.