Project description:Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologues are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X-chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodelling and silencing fails, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1, and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis and meiotic chromosome silencing.
Project description:The switch from mitosis to meiosis is a major transition that takes place during germ cell development. The precise sequence and the different gene expression programs activated during this process are only partly known. Here, we applied single-cell mRNA sequencing to interrogate the transcriptional changes that occur during the early steps of male germ cell differentiation. We isolated single cells from testes using a Dazl-GFP reporter mouse, which allowed us to focus on germ cells undergoing the mitotic to meiotic transition. We identified 4 distinct meiotic stages with unique transcriptome profiles and reconstructed the timeline of the meiotic entry in silico, from spermatogonia up to the pachytene stage, identifying transcriptional changes with an unprecedented resolution. This allowed us to characterize 3 major transitions in the meiotic prophase 1 of the male germline: meiotic entry, the meiotic sex chromosome inactivation (MSCI), and concomitant pachytene transcriptional activation. Meiotic entry is initiated following the downregulation of a tightly connected set of pluripotency factors and accompanied by a global transcriptional silencing. In contrast, during subsequent sex chromosome inactivation at the zygotene stage, gene silencing proceeds in a defined order, related to gene function.
Project description:Meiotic sex chromosome inactivation (MSCI) is an essential process in the male germline. While genetic experiments have established that the DNA damage response (DDR) pathway directs MSCI, due to limitations to the experimental systems available, mechanisms underlying MSCI remain largely unknown. Here we establish a system to study MSCI ex vivo, based on a short-term culture method, and demonstrate that active DDR signaling is required both to initiate and maintain MSCI via a dynamic and reversible process. DDR-directed MSCI follows two layers of modifications: active DDR-dependent reversible processes and irreversible histone post-translational modifications. Further, the DDR initiates MSCI independent of the downstream repressive histone mark H3K9 trimethylation (H3K9me3), thereby demonstrating that active DDR signaling is the primary mechanism of silencing in MSCI. By unveiling the dynamic nature of MSCI, and its governance by active DDR signals, our study highlights the sex chromosomes as an active signaling hub in meiosis.
Project description:During spermatogenesis, germ cells that fail to synapse their chromosomes or fail to undergo meiotic sex chromosome inactivation (MSCI) are eliminated via apoptosis during mid-pachytene. Previous work showed that Y-linked genes Zfy1 and Zfy2 act as “executioners” for this checkpoint, and that wrongful expression of either gene during pachytene triggers germ cell death. Here, we show that in mice, Zfy genes are also necessary for efficient MSCI and the sex chromosomes are not correctly silenced in Zfy-deficient spermatocytes. In addition to this microarray data set, qPCR and RNA FISH data were used to confirm that MSCI is leaky in males lacking Zfy genes. The array data demonstrates that transgenic restoration of Zfy2 corrects the MSCI leakage phenotype. The associated RNA FISH data demonstrates that Zfy1 and Zfy2 are both able to correct the leakage phenotype.
Project description:Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization, and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 down-regulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a novel meiotic and embryonic competence factor in meiosis and mitosis, safeguarding genome integrity at the onset of life. We performed expression profiling on pools of 16 denuded GV-oocytes isolated per mouse. We used oocytes from 4 Setdb1 f/+; Zp3-cre mice and 2 Setdb1 f/- mice as controls and oocytes from 4 Setdb1 f/-; Zp3-cre mice as mutant.
Project description:Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization, and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 down-regulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a novel meiotic and embryonic competence factor in meiosis and mitosis, safeguarding genome integrity at the onset of life.
Project description:Meiotic recombination differs between males and females, however, when and how these differences are established is unknown. We identify extensive sex differences at recombination initiation by mapping hotspots of meiotic DNA double strand breaks in male and female mice. Contrary to past findings in humans, few hotspots are used uniquely in either sex. Instead, grossly different recombination landscapes result from up to 15-fold differences in hotspot use between males and females. Indeed, most recombination occurs at sex-biased hotspots. Sex biased hotspots appear to be partly determined by chromosome structure, and DNA methylation, absent in females at the onset of meiosis, plays a substantial role. Sex differences are also evident later in meiosis as the repair frequency of distal meiotic breaks as crossovers diverges in males and females. Suppression of distal crossovers may help to minimize age-related aneuploidy that arises due to cohesion loss during dictyate arrest in females.