Institut de recherche biomédicale
Institut de recherche biomédicale
Project leader
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Julie COCQUET +33 144412312 |
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• 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)
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).
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Figure: From Moretti et al. 2017. Model presenting the mechanism by which SLY controls gene expression and chromatin remodeling during sperm differentiation. In WT round spermatids (left panel), SLY (in blue) interacts with the SMRT/N-CoR complex (which comprises TBL1XR1, TBL1X, NCOR1 and HDAC3) and is located at the start of genes involved in gene regulation, chromatin regulation and the ubiquitin pathway. In particular, SLY directly controls the expression of X-chromosome-encoded genes coding for H2.A variants (such as H2A.B3) and of the H3K79 methyltransferase DOT1L. In elongating spermatids, there is a wave of H3K79 dimethylation (orange circles) and of histone H4 acetylation (green circles); those modifications are expected to be a prerequisite to the efficient removal of nucleosomes (light pink oval) and replacement by protamines (purple oval), a process which is required to achieve optimal compaction of the spermatozoa nucleus. When SLY is knocked down (right panel), X-encoded H2.A variants are upregulated and more incorporated in the spermatid chromatin, while DOT1L is downregulated. DOT1L downregulation leads to a decrease in dimethylated H3K79 and acetylated histone H4 in elongating spermatids. Alterations in the spermatid chromatin structure affect the replacement of nucleosomes by protamines and lead to a higher proportion of nucleosomes and a decreased proportion of protamines. As a result, Sly-deficient spermatozoa are abnormally shaped, less compact and present a higher susceptibility to DNA damage than WT spermatozoa |
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.
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Figure from Moretti et al 2020. |
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