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Epigenetic decisions and reproduction in mammals

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Group leader : Déborah Bourc'his
Epigenetic decisions and reproduction in mammals

Keywords

Epigenetics, DNA methylation, germ line, mouse development, reproduction, genomic imprinting

Plain

Our research is centered on the window of conception in mammals. We are interested in the epigenetic information, beyond the genetic material, that prepares the egg and the sperm for the process of fertilization and how this information impacts immediately on the embryo but also, on a longer term, on the adult individual and eventually, on the next generations.

 

The process of germ line differentiation allows the production of mature gametes that carry both genetic  and epigenetic information.

Epigenetic remodelling of the germ line has a dual effect, creating a state of extreme differentiation that allows the egg and the sperm to perform fertilization, while maintaining pluripotent properties necessary to support the development of a new individual.


 

Mammals have specifically evolved a DNA methylation-based mechanism of gametic programming. Gametic methylation patterns have immediate effects on gametic identity, genome integrity through the protection against mobile genetic elements, and sex-specific differentiation of male and female gametes. They also have long-term consequences on the development of the progeny after fertilization. We are interested in understanding how DNA methylation patterns are shaped and how they provide the gametes with the potential to promote an entire new organism and faithfully perpetuate the hereditary material. We combine genetic, cellular and developmental approaches with genome-wide and biochemical strategies, and rely mouse models of deficiency in DNA methylation and other epigenetic modifications. We are also extending our knowledge to human through collaborations with fertility centers.

Our work can be subdivided in three themes related to the epigenetic programming of the germ line and its influence on reproduction.

  • The first focuses on the protection of the male germ line against transposons. These genetic parasites make up the majority of the mammalian genomes and can impact on gene expression and on genome integrity in a variety of ways. We are investigating the cellular and genetic damages that transposons inflict on the male germ line, and the mechanisms developed by the germ line to counteract these effects.
  • The second aspect deals with the importance of epigenetic processes in the female germ line. We have been developing genome-wide methylation screening for the identification of genes specifically methylated in the oocyte. Oocyte methylation is particularly involved in the control of parent-specific expression of imprinted genes. Imprinted genes greatly impacts on embryonic development but their exact number is still elusive. Using our mutant models, we have been developing a genome-wide methylation screening for the identification of new imprinted genes. We are also screening for new targets of oocyte methylation, and in particular for genes that are epigenetically programmed in the oocyte for their participation to embryonic development after fertilization.
  • Finally, in an attempt to understand the uniqueness of the germ line material, we are comparing the mechanisms underlying the acquisition of DNA methylation during gametogenesis versus early embryogenesis, two periods that are subject to intensive epigenetic changes.
Correct DNA methylation patterns are paramount for the generation of functional gametes capable of forming viable offspring, but also for the regulation of pluripotency states and the maintenance of genome architecture and expression in somatic cells. Our work, not only impacts on the field of reproduction and development, but also on stem cell biology and cancer.
 Fig.1: Testis and ovary showing Dnmt3L-expressing germ cells (blue) at the time of acquisition of gametic DNA methylation patterns.Fig.1: Testis and ovary showing Dnmt3L-expressing germ cells (blue) at the time of acquisition of gametic DNA methylation patterns.Fig.2: Reactivation and accumulation of transposons in methylation-deficient male germ cells (red: synaptonemal complex, green: ORF1 proteins produced by LINE-1 transposons).Fig.2: Reactivation and accumulation of transposons in methylation-deficient male germ cells (red: synaptonemal complex, green: ORF1 proteins produced by LINE-1 transposons).

Fig.3: Prenatal development of the mouse, at 10, 12 and 17 days of gestationFig.3: Prenatal development of the mouse, at 10, 12 and 17 days of gestation

   

  
   

  

Key publications

  • Year of publication : 2016

  • DNA methylation is extensively remodeled during mammalian gametogenesis and embryogenesis. Most transposons become hypomethylated, raising the question of their regulation in the absence of DNA methylation. To reproduce a rapid and extensive demethylation, we subjected mouse ES cells to chemically defined hypomethylating culture conditions. Surprisingly, we observed two phases of transposon regulation. After an initial burst of de-repression, various transposon families were efficiently re-silenced. This was accompanied by a reconfiguration of the repressive chromatin landscape: while H3K9me3 was stable, H3K9me2 globally disappeared and H3K27me3 accumulated at transposons. Interestingly, we observed that H3K9me3 and H3K27me3 occupy different transposon families or different territories within the same family, defining three functional categories of adaptive chromatin responses to DNA methylation loss. Our work highlights that H3K9me3 and, most importantly, polycomb-mediated H3K27me3 chromatin pathways can secure the control of a large spectrum of transposons in periods of intense DNA methylation change, ensuring longstanding genome stability.
  • Year of publication : 2015

  • DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L(-/-) meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events.
  • Year of publication : 2014

  • Many loci maintain parent-of-origin DNA methylation only briefly after fertilization during mammalian development: Whether this form of transient genomic imprinting can impact the early embryonic transcriptome or even have life-long consequences on genome regulation and possibly phenotypes is currently unknown. Here, we report a maternal germline differentially methylated region (DMR) at the mouse Gpr1/Zdbf2 (DBF-type zinc finger-containing protein 2) locus, which controls the paternal-specific expression of long isoforms of Zdbf2 (Liz) in the early embryo. This DMR loses parental specificity by gain of DNA methylation at implantation in the embryo but is maintained in extraembryonic tissues. As a consequence of this transient, tissue-specific maternal imprinting, Liz expression is restricted to the pluripotent embryo, extraembryonic tissues, and pluripotent male germ cells. We found that Liz potentially functions as both Zdbf2-coding RNA and cis-regulatory RNA. Importantly, Liz-mediated events allow a switch from maternal to paternal imprinted DNA methylation and from Liz to canonical Zdbf2 promoter use during embryonic differentiation, which are stably maintained through somatic life and conserved in humans. The Gpr1/Zdbf2 locus lacks classical imprinting histone modifications, but analysis of mutant embryonic stem cells reveals fine-tuned regulation of Zdbf2 dosage through DNA and H3K27 methylation interplay. Together, our work underlines the developmental and evolutionary need to ensure proper Liz/Zdbf2 dosage as a driving force for dynamic genomic imprinting at the Gpr1/Zdbf2 locus.
  • Year of publication : 2013

  • A stimulatory DNA methyltransferase co-factor, Dnmt3L, has evolved in mammals to assist the process of de novo methylation, as genetically demonstrated in the germline. The function of Dnmt3L in the early embryo remains unresolved. By combining developmental and genetic approaches, we find that mouse embryos begin development with a maternal store of Dnmt3L, which is rapidly degraded and does not participate in embryonic de novo methylation. A zygotic-specific promoter of Dnmt3l is activated following gametic methylation loss and the potential recruitment of pluripotency factors just before implantation. Importantly, we find that zygotic Dnmt3L deficiency slows down the rate of de novo methylation in the embryo by affecting methylation density at some, but not all, genomic sequences. Dnmt3L is not strictly required, however, as methylation patterns are eventually established in its absence, in the context of increased Dnmt3A protein availability. This study proves that the postimplantation embryo is more plastic than the germline in terms of DNA methylation mechanistic choices and, importantly, that de novo methylation can be achieved in vivo without Dnmt3L.
  • Year of publication : 2012

  • Identifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life.
  • Year of publication : 2010

  • Transient populations of cis- and trans-acting small RNAs have recently emerged as key regulators of extensive epigenetic changes taking place during periconception, which encompasses gametogenesis, fertilization, and early zygotic development. These small RNAs are not only important to maintain genome integrity in the gametes and zygote, but they also actively contribute to assessing the compatibility of parental genomes at fertilization and to promoting long-term memory of the zygotic epigenetic landscape by affecting chromatin. Striking parallels exist in the biogenesis and modus operandi of these molecules among diverse taxa, unraveling universal themes of small-RNA-mediated epigenetic reprogramming during sexual reproduction.
  • In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
  • Retrotransposable elements comprise around 50% of the mammalian genome. Their activity represents a constant threat to the host and has prompted the development of adaptive control mechanisms to protect genome architecture and function. To ensure their propagation, retrotransposons have to mobilize in cells destined for the next generation. Accordingly, these elements are particularly well suited to transcriptional networks associated with pluripotent and germinal states in mammals. The relaxation of epigenetic control that occurs in the early developing germline constitutes a dangerous window in which retrotransposons can escape from host restraint and massively expand. What could be observed as risky behavior may turn out to be an insidious strategy developed by germ cells to sense retrotransposons and hold them back in check. Herein, we review recent insights that have provided a detailed picture of the defense mechanisms that concur toward retrotransposon silencing in mammalian genomes, and in particular in the germline. In this lineage, retrotransposons are hit at multiple stages of their life cycle, through transcriptional repression, RNA degradation and translational control. An organized cross-talk between PIWI-interacting small RNAs (piRNAs) and various nuclear and cytoplasmic accessories provides this potent and multi-layered response to retrotransposon unleashing in early germ cells.