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    (Epi)genetic regulation during spermatogenesis

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    Project leader


    Participants

        •    Julie Cocquet, Principal investigator (CR1 INSERM)
        •    Melina Blanco (PhD student)
        •    Manon Coulée (PhD student)
        •    Laila El Khattabi (MCU-PH, Cochin Hospital staff)
        •    Clara Gobé (postdoctoral research fellow)
        •    Côme Ialy-Radio (AI INSERM)

     

    Main research Topics

     

    (1) Gene regulation and chromatin remodeling during spermatogenesis

    Approximately one man out of ten suffers from infertility, with a spermatogenesis defect in many cases. The cause, whether genetic or environmental, remains often unknown, with ~75% of male infertility defined as ‘idiopathic’.
    Spermatogenesis, the process by which spermatozoa develop from immature germ cells in the seminiferous tubules of the testis, is generally divided in three steps: proliferation of spermatogonial stem cells, meiosis, and differentiation of haploid germ cells, known as spermatids, into spermatozoa. Our work is mainly focused on this last step, called spermiogenesis. It is a fascinating process in term of gene expression dynamics and chromatin remodeling (Blanco & Cocquet, 2019). During this step, spermatids undergo profound morphological and functional changes to become spermatozoa. They acquire their specific shape through the loss of most of their cytoplasm, the biogenesis of a flagellum and an acrosome, and the compaction of their nucleus to ~1/5th of its original size. Many of the morphological and functional changes occurring during sperm differentiation are orchestrated by the spermatid (epi)genetic program, with thousands of genes highly expressed in spermatids, before transcription shuts down due to chromatin compaction. A significant proportion of spermatid-specific genes is located on the X and Y chromosomes and appears to be co-regulated.

    We study spermatogenesis at the gene and the chromatin level using omics, in vivo and in vitro approaches, to identify and characterize novel regulators/pathways required for sperm differentiation and male fertility [see below, and (Crespo et al. 2020)].

    Our project also aims at studying the impact of spermatozoa epigenetic program on reproductive efficiency, embryo development and progeny’s health. Indeed it is now well-known that, upon fertilization, the sperm cell contributes to the embryo with more than its DNA (Champroux et al 2018). Epigenetic information (chromatin, RNA molecules, DNA modifications, etc.) is also transmitted to the embryo and could affect embryo development and offspring health, in case of deregulation. It is therefore essential to better understand the (epi)genetic processes occurring during sperm differentiation

    Results

    In the last decade, we have extensively studied two multicopy genes, named Slx/Slxl1 and Sly, which are respectively encoded by the mouse X and Y chromosomes. We have shown that absence/knock-down of Sly leads to a wide range of spermiogenesis defects, such as deformed spermheads, reduced motility, abnormal chromatin compaction and DNA damage, which cause male infertility. Those defects are associated with deregulation of hundreds of sex chromosome-encoded genes, as well as a smaller portion of autosomal genes (such as the genes of the Speer family) (Cocquet et al. PLos Biol 2009; Riel et al. J Cell Sci 2013; Moretti et al. Cell Death & Diff 2017 ). Interestingly, we have observed that Slx/Slxl1, the X-linked homologs of Sly, have a role opposite to that of Sly on gene expression (Cocquet et al. PLoS Genet 2012). Slx/Slxl1 deficiency also leads to sperm differentiation defects characterized by abnormal sperm morphology and increased spermatid apoptosis (Cocquet et al. Mol Biol Cell 2010). Using Slx/Slxl1 and Sly as entry points, we have identified novel actors of gene/chromatin regulation during spermiogenesis. By ChIP seq, we have shown that SLY protein is enriched at the promoter of thousands of genes involved in gene expression, chromatin regulation, and the ubiquitin pathway. Among those genes is Dot1l, which encodes the only known H3K79 methyltransferase; both Dot1l expression and H3K79 methylation level are downregulated Sly-deficient in spermatids. In parallel, we have used a global approach (co-immunoprecipitation followed by mass spectrometry) to identify novel SLY protein partners, and found that SLY interacts with SMRT/NcoR, a protein complex involved in transcriptional regulation, and which appears to be relevant to gene regulation during spermiogenesis (Moretti et al. Cell Death & Diff 2017).


     

    (2) Molecular mechanism of the genomic conflict between the mouse sex chromosomes - competition between Slx/Slxl1 and Sly

    While studying the molecular roles of Slx/Slxl1 and Sly, we discovered that they are involved in an intragenomic conflict that causes segregation distortion in addition to male infertility. Indeed, Slx/Slxl1 and Sly are "selfish" genes – each promoting its own transmission to the detriment of the other. Slx/Slxl1 and Sly genes are present dozens of times on the mouse X and Y chromosomes, respectively. Their number of copies is not the same depending on the species and has co-evolved during rodent evolution. The “conflict” for transmission in which Slx/Slxl1 and Sly are engaged led to a genetic arms race in which the amplification of the number of copies of one is counterbalanced by the amplification of the other. An imbalance in the number of copies of one versus the other (obtained by genetic mutants inducing a "knock-down" of Slx/Slxl1 or Sly) produces a skewed sex ratio of the progeny associated with hypofertility, or infertility, if copy number ratio is severely imbalanced. The effect of these two genes compensates each other regardless of their level of expression since the double "knockdown" of Slx/Slxl1 and Sly improves the phenotype of the simple "knockdown" of one or the other (Cocquet et al. PLoS Genet 2012).

    Our project aims at studying the competition at the molecular level and identifying other actors of the conflict.

    Results

    We have recently shown that SLX/SLXL1 and SLY proteins are present at the promoter of thousands of spermatid genes, and that the knockdown of Sly increases the presence of SLX/SLXL1 at these promoters (Moretti et al. MBE 2020 ). Competition at the promoter level is mediated via SSTY protein, with which SLX/SLXL1 or SLY (but not the 2 together) interacts. Interestingly, SSTY belongs to a family of proteins, SPINDLIN, which recognizes the chromatin mark H3K4me3, characteristic of the promoter of active (expressed) genes. When SLY predominates, the genes with which it is associated are under-expressed, conversely when SLX/SLXL1 predominates these genes are over-expressed. To complete the elucidation of the molecular mechanism, we found that SLY, but not SLX/SLXL1, interacts with proteins of the SMRT/N-Cor complex that repress transcription; in case of Sly knockdown, this complex is less recruited at the promoters of SLX/SLY target genes resulting in upregulation. This result explains, at least in part, the fact that SLY acts as a repressor and SLX/SLXL1, as an activator.

    From an evolutionary point of view, it is important to note that Ssty gene is itself multicopy and carried by the Y chromosome. Besides, many of SLX/SLXL1, SLY and SSTY target genes are also multicopy genes that were co-amplified during Muroid evolution, particularly Speer/Takusan genes amplified >150 times on chromosome 14.

    Our work is an important step towards elucidating the molecular mechanism behind the competition between X and Y genes - a genomic conflict that has influenced genome organization and evolution of several rodents. This type of phenomenon is predicted to be quite widespread, and create hybrid incompatibility, lineage divergence and eventually speciation.

     

    Selected publications

    • Moretti C, Blanco M, Ialy-Radio C, Serrentino ME, Gobé C, Friedman R, Battail C, Leduc M, Ward MA, Vaiman D, Tores F, Cocquet J. Battle of the sex chromosomes: competition between X- and Y-chromosome encoded proteins for partner interaction and chromatin occupancy drives multi-copy gene expression and evolution in muroid rodents. Mol Biol Evol. 2020 Jul 13:msaa175. doi: 10.1093/molbev/msaa175. Online ahead of print. PMID: 32658962
    • Crespo M, Damont A, Blanco M, Lastrucci E, Kennani SE, Ialy-Radio C, Khattabi LE, Terrier S, Louwagie M, Kieffer-Jaquinod S, Hesse AM, Bruley C, Chantalat S, Govin J, Fenaille F, Battail C, Cocquet J, Pflieger D. Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes. Nucleic Acids Res. 2020 May 7;48(8):4115-4138. doi: 10.1093/nar/gkaa163. PMID: 32182340
    • Blanco M, Cocquet J. Genetic Factors Affecting Sperm Chromatin Structure. Adv Exp Med Biol. 2019;1166:1-28.
    • Riel JM, Yamauchi Y, Ruthig VA, Malinta QU, Blanco M, Moretti C, Cocquet J, Ward MA. Rescue of Sly Expression Is Not Sufficient to Rescue Spermiogenic Phenotype of Mice with Deletions of Y Chromosome Long Arm. Genes (Basel). 2019 Feb 12;10(2).
    • Champroux A, Cocquet J, Henry-Berger J, Drevet JR, Kocer A. A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance. Front Cell Dev Biol. 2018 May 15;6:50.
    • El Kennani S, Adrait A, Permiakova O, Hesse AM, Ialy-Radio C, Ferro M, Brun V, Cocquet J, Govin J, Pflieger D.(2018) Systematic quantitative analysis of H2A and H2B variants by targeted proteomics. Epigenetics Chromatin. 2018 Jan 12;11(1):2.
    • Moretti C*, Serrentino ME*, Ialy-Radio C, Delessard M, Soboleva TA, Tores F, Leduc M, Nitschké P, Drevet JR, Tremethick DJ, Vaiman D, Kocer A, Cocquet J. SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation. Cell Death Differ. 2017 24:1029-1044
    • Moretti C, Vaiman D, Tores F, Cocquet J. Expression and epigenomic landscape of the sex chromosomes in mouse post-meiotic male germ cells. Epigenetics Chromatin. 2016 Oct 27;9:47. eCollection 2016
    • Comptour A*, Moretti C*, Serrentino ME, Auer J, Ialy-Radio C, Ward MA, Touré A, Vaiman D, Cocquet J. SSTY proteins co-localize with the post-meiotic sex chromatin and interact with regulators of its expression. FEBS J. 2014 281: 1571-84
    • Riel JM, Yamauchi Y, Sugawara A, Li HY, Ruthig V, Stoytcheva Z, Ellis PJ, Cocquet J, Ward MA. (2013) Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging. J Cell Sci 126: 803-813
    • Cocquet J, Ellis PJ, Mahadevaiah SK, Affara NA, Vaiman D, Burgoyne P. (2012) A genetic basis for a postmeiotic X versus Y chromosome intragenomic conflict in the mouse. PLoS Genet 8: e1002900.
    • Cocquet J, Ellis PJ, Yamauchi Y, Riel JM, Karacs TP, Rattigan A, Ojarikre OA, Affara NA, Ward MA, Burgoyne PS. (2010) Deficiency in the multicopy Sycp3-like X-linked genes Slx and Slxl1 causes major defects in spermatid differentiation. Mol Biol Cell. 21:3497-505.
    • Cocquet J, Ellis PJ, Yamauchi Y, Mahadevaiah SK, Affara NA, Ward MA, Burgoyne P. (2009) The multicopy gene Sly represses the sex chromosomes in the male mouse germline after meiosis. PLoS Biol 7: e1000244.

     

    Funding