RESUMO
H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.
Assuntos
Heterocromatina , Histonas , Masculino , Células Germinativas/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Meiose/genética , Metilação , Animais , CamundongosRESUMO
To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.
Assuntos
Regulação da Expressão Gênica , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , TranscriptomaRESUMO
H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.
RESUMO
Vascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. The gene-regulatory mechanisms underpinning endothelial specialization are largely unknown, as are the roles of chromatin organization in regulating endothelial cell transcription. To investigate the relationships between chromatin organization and gene expression, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-sequencing assays at specific time points. Long-range intrachromosomal contacts increase over the course of differentiation, accompanied by widespread heteroeuchromatic compartment transitions that are tightly associated with transcription. Dynamic topologically associating domain boundaries strengthen and converge on an endothelial cell state, and function to regulate gene expression. Chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes essential to vascular biology. Chromatin dynamics guide transcription in endothelial cell development and promote the divergence of endothelial cells from cardiomyocytes.
Assuntos
Cromatina , Células Endoteliais , Humanos , Diferenciação Celular/genética , Regulação da Expressão GênicaRESUMO
In mammalian male meiosis, the heterologous X and Y chromosomes remain unsynapsed and, as a result, are subject to meiotic sex chromosome inactivation (MSCI). MSCI is required for the successful completion of spermatogenesis. Following the initiation of MSCI, the X and Y chromosomes undergo various epigenetic modifications and are transformed into a nuclear body termed the XY body. Here, we review the mechanisms underlying the initiation of two essential, sequential processes in meiotic prophase I: MSCI and XY-body formation. The initiation of MSCI is directed by the action of DNA damage response (DDR) pathways; downstream of the DDR, unique epigenetic states are established, leading to the formation of the XY body. Accumulating evidence suggests that MSCI and subsequent XY-body formation may be driven by phase separation, a physical process that governs the formation of membraneless organelles and other biomolecular condensates. Thus, here we gather literature-based evidence to explore a phase separation hypothesis for the initiation of MSCI and the formation of the XY body.
Assuntos
Mecanismo Genético de Compensação de Dose , Meiose , Modelos Biológicos , Cromossomos Sexuais/metabolismo , Animais , Dano ao DNA/genética , Reparo do DNA/genética , Humanos , Meiose/genéticaRESUMO
Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.
Assuntos
Retrovirus Endógenos/genética , Espermatogênese/genética , Espermatozoides/fisiologia , Animais , Cromatina/genética , Cromatina/virologia , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Humanos , Elementos Nucleotídeos Longos e Dispersos , Masculino , Mamíferos/genética , Mamíferos/virologia , Meiose , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitose , Mutação , Proteínas Proto-Oncogênicas c-myb/genética , Sequências Repetitivas de Ácido Nucleico , Roedores/genética , Roedores/virologia , Espermatozoides/virologia , Transativadores/genética , TranscriptomaRESUMO
Owing to bursts in the expression of thousands of germline-specific genes, the testis has the most diverse and complex transcriptome of all organs. By analyzing the male germline of mice, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts in germline gene expression after the mitosis-to-meiosis transition. SE reorganization is regulated by two molecular events: the establishment of meiosis-specific SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and the resolution of SEs in mitotically proliferating cells via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Before entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia to ensure the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotic proliferation phases.
Assuntos
Elementos Facilitadores Genéticos , Meiose/genética , Mitose/genética , Proteínas do Grupo Polycomb/genética , Proteínas Proto-Oncogênicas c-myb/genética , Espermatogênese/genética , Transativadores/genética , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteínas do Grupo Polycomb/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Espermatogônias/citologia , Espermatogônias/fisiologia , Transativadores/metabolismo , Cromossomo X/genéticaRESUMO
To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.
Assuntos
Meiose/fisiologia , Recombinação Genética/fisiologia , Animais , DNA Helicases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismoRESUMO
BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.
Assuntos
Instabilidade Genômica , Células Germinativas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebra Cromossômica , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Masculino , Meiose , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Espermatócitos/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatogênese/efeitos dos fármacos , Complexo Sinaptonêmico/efeitos dos fármacos , Complexo Sinaptonêmico/metabolismo , Tamoxifeno/farmacologia , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.
Assuntos
Dano ao DNA , Mecanismo Genético de Compensação de Dose , Meiose , Cromossomos Sexuais/genética , Transdução de Sinais , Espermatogênese/genética , Animais , Histonas/metabolismo , Masculino , Camundongos , FosforilaçãoRESUMO
Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Meiose/fisiologia , Espermátides/crescimento & desenvolvimento , Espermatócitos/crescimento & desenvolvimento , Espermatogênese/fisiologia , Animais , Cromatina/metabolismo , Cromossomos/metabolismo , Interfase/fisiologia , Masculino , Prófase Meiótica I/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos/fisiologiaRESUMO
Genes involved in sexual reproduction diverge rapidly as a result of reproductive fitness. Here, we identify a novel protein domain in the germline-specific Polycomb protein SCML2 that is required for the establishment of unique gene expression programs after the mitosis-to-meiosis transition in spermatogenesis. We term this novel domain, which is comprised of rapidly evolved, DNA-binding repeat units of 28 amino acids, the SCML2 DNA-binding (SDB) repeats. These repeats are acquired in a specific subgroup of the rodent lineage, having been subjected to positive selection in the course of evolution. Mouse SCML2 has two DNA-binding domains: one is the SDB repeats and the other is an RNA-binding region, which is conserved in human SCML2. For the recruitment of SCML2 to target loci, the SDB repeats cooperate with the other functional domains of SCML2 to bind chromatin. The cooperative action of these domains enables SCML2 to sense DNA hypomethylation in an in vivo chromatin environment, thereby enabling SCML2 to bind to hypomethylated chromatin. We propose that the rapid evolution of SCML2 is due to reproductive adaptation, which has promoted species-specific gene expression programs in spermatogenesis.
Assuntos
Evolução Molecular , Proteínas do Grupo Polycomb/genética , Animais , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Filogenia , Ligação Proteica , Domínios Proteicos , Proteínas RecombinantesRESUMO
The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry- mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry- females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry- PGCs was associated with aberrant ß-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry- oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as "Swyer syndrome," in which patients with an XY karyotype present as typical females and are infertile.
Assuntos
Disgenesia Gonadal 46 XY/fisiopatologia , Oócitos/crescimento & desenvolvimento , Proteína da Região Y Determinante do Sexo/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo Y , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitose , Mutação , TranscriptomaRESUMO
A distinct form of X chromosome inactivation takes place during male meiosis, when the male sex chromosomes undergo a phenomenon known as meiotic sex chromosome inactivation (MSCI). MSCI is directed by DNA damage response signaling independent of Xist RNA to silence the transcriptional activity of the sex chromosomes, an essential event in male germ cell development. Here, we present protocols for the preparation and analyses of chromosome spread slides of mouse meiotic spermatocytes, thereby enabling a quick, inexpensive, and powerful cytological method to complement gene expression studies.
Assuntos
Imunofluorescência/métodos , Meiose , Cromossomos Sexuais , Espermatócitos/metabolismo , Inativação do Cromossomo X , Animais , Epigenômica/métodos , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Espermatócitos/citologiaRESUMO
Spermatogenesis involves the progressive reorganization of heterochromatin. However, the mechanisms that underlie the dynamic remodeling of heterochromatin remain unknown. Here, we identify SCML2, a germline-specific Polycomb protein, as a critical regulator of heterochromatin organization in spermatogenesis. We show that SCML2 accumulates on pericentromeric heterochromatin (PCH) in male germ cells, where it suppresses PRC1-mediated monoubiquitylation of histone H2A at Lysine 119 (H2AK119ub) and promotes deposition of PRC2-mediated H3K27me3 during meiosis. In postmeiotic spermatids, SCML2 is required for heterochromatin organization, and the loss of SCML2 leads to the formation of ectopic patches of facultative heterochromatin. Our data suggest that, in the absence of SCML2, the ectopic expression of somatic lamins drives this process. Furthermore, the centromere protein CENP-V is a specific marker of PCH in postmeiotic spermatids, and SCML2 is required for CENP-V localization on PCH. Given the essential functions of PRC1 and PRC2 for genome-wide gene expression in spermatogenesis, our data suggest that heterochromatin organization and spermatogenesis-specific gene expression are functionally linked. We propose that SCML2 coordinates the organization of heterochromatin and gene expression through the regulation of Polycomb complexes.
Assuntos
Heterocromatina/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Espermátides/metabolismo , Espermatogênese , Animais , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Masculino , Meiose , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Espermátides/citologiaRESUMO
Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.
Assuntos
Histonas/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Espermatogênese/fisiologia , Espermatogônias/metabolismo , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas do Grupo Polycomb/genética , Espermatogônias/citologiaRESUMO
The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids.
Assuntos
Histonas/metabolismo , Proteínas do Grupo Polycomb/fisiologia , Cromossomos Sexuais/genética , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação/genética , Acetilação , Animais , Feminino , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cromossomos Sexuais/metabolismo , Espermátides/fisiologia , Espermatogênese/genéticaRESUMO
The continuity of life depends on mechanisms in the germline that ensure the integrity of the genome. The DNA damage response/checkpoint kinases ATM and ATR are essential signaling factors in the germline. However, it remains unknown how a downstream transducer, Checkpoint Kinase 1 (CHEK1 or CHK1), mediates signaling in the male germline. Here, we show that CHEK1 has distinct functions in both the mitotic and meiotic phases of the male germline in mice. In the mitotic phase, CHEK1 is required for the resumption of prospermatogonia proliferation after birth and the maintenance of spermatogonia. In the meiotic phase, we uncovered two functions for CHEK1: one is the stage-specific attenuation of DNA damage signaling on autosomes, and the other is coordination of meiotic stage progression. On autosomes, the loss of CHEK1 delays the removal of DNA damage signaling that manifests as phosphorylation of histone variant H2AX at serine 139 (γH2AX). Importantly, CHEK1 does not have a direct function in meiotic sex chromosome inactivation (MSCI), an essential event in male meiosis, in which ATR is a key regulator. Thus, the functions of ATR and CHEK1 are uncoupled in MSCI, in contrast to their roles in DNA damage signaling in somatic cells. Our study reveals stage-speciï¬c functions for CHEK1 that ensure the integrity of the male germline.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Meiose , Transdução de Sinais , Espermatogônias/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/genética , Masculino , Camundongos , Camundongos Knockout , Espermatogônias/citologiaRESUMO
During spermatogenesis, germ cells undergo massive cellular reconstruction and dynamic chromatin remodeling to facilitate highly diverse transcriptomes, which are required for the production of functional sperm. However, it remains unknown how germline chromatin is organized to promote the dynamic, complex transcriptomes of spermatogenesis. Here, using ATAC-seq, we establish the varied landscape of open chromatin during spermatogenesis. We identify the reorganization of accessible chromatin in intergenic and intronic regions during the mitosis-to-meiosis transition. During the transition, mitotic-type open chromatin is closed while the de novo formation of meiotic-type open chromatin takes place. Contrastingly, differentiation processes such as spermatogonial differentiation and the meiosis-to-postmeiosis transition involve chromatin closure without the de novo formation of accessible chromatin. In spermiogenesis, the germline-specific Polycomb protein SCML2 promotes the closure of open chromatin at autosomes for gene suppression. Paradoxically, we identify the massive de novo formation of accessible chromatin when the sex chromosomes undergo meiotic sex chromosome inactivation, and this is also mediated by SCML2. These results reveal meiotic sex chromosome inactivation as an active process for chromatin organization. Together, our results unravel the genome-wide, dynamic reorganization of open chromatin and reveal mechanisms that underlie diverse transcriptomes during spermatogenesis.