Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells

In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells

E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific

peer-reviewedBackground: Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species.Background: Spermatozoa have a remarkable epigenResults: The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. Conclusions: These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis

Identification of an imprinted gene network and its role in controlling transitions between proliferation, quiescence and differentiation.

L'empreinte génomique parentale est un mécanisme de régulation épigénétique conduisant à la répression d'un allèle d'un gène en fonction de son origine parentale. Ce mécanisme affecte un nombre restreint de gènes chez les mammifères métathériens et euthériens. Ces gènes, dits gènes soumis à empreinte (GSE), ont des fonctions moléculaires variées et sans lien apparent. Cependant, deux thèmes reviennent de manière récurrente dans leurs fonctions: le contrôle de la croissance embryonnaire et la tumorigenèse. Ma thèse a consisté à démontrer l'existence d'un lien fonctionnel entre les GSE. Nous montrons que les GSE s'inscrivent dans un même réseau de co-expression transcriptionnelle et qu'ils sont co-régulés dans différentes situations biologiques lors des transitions entre les différents états cellulaires. En effet, une induction coordonnée de la plupart des GSE a lieu lors des sorties du cycle cellulaire, réversibles (quiescence) ou non (différenciation). Les perturbations individuelles de l'expression de plusieurs GSE dans le modèle des pré-adipocytes 3T3-L1 confirment un rôle du réseau des GSE dans le contrôle des transitions entre prolifération, quiescence et différenciation. De plus, l'analyse des gènes bi-alléliques inclus dans le même réseau de co-régulation que les GSE montre un enrichissement en gènes de la matrice extracellulaire. La fonction associée à ce réseau serait donc le contrôle des transitions entre les différents états cellulaires, via le remodelage de la matrice extracellulaire. Pour conclure, outre l'identification d'une fonction commune aux GSE, nos résultats suggèrent un scénario pour le ciblage de ces gènes par l'empreinte génomique parentale au cours de l'évolution des mammifères.Genomic imprinting is an epigenetic mechanism leading to the repression of one allele of a gene, depending on its parental origin. This mechanism affects a small number of genes in metatherian and eutherian mammals. These genes, named imprinted genes (IGs), display various molecular functions and thus seem unrelated. However, their alterations are frequently associated with the control of embryonic growth and tumorigenesis. My PhD project has consisted in demonstrating a functional link between IGs. We show that IGs are frequently co-expressed and belong to a common gene network. They are co-regulated in biological situations corresponding to the transitions between different cellular states. Coordinated induction of most IGs takes place at the outputs of the cell cycle. Loss and gain of function experiments of several IGs in the 3T3-L1 pre-adipocyte model demonstrate a role of the IG network in controlling transitions between cellular states (proliferation, quiescence and differentiation). In addition to IGs, this network also includes bi-allelic genes, with many extracellular matrix genes. Therefore, the function associated with the IG network could be the fine control of transitions between cellular states through a remodeling of the extracellular matrix.To conclude, in addition to the identification of a common cellular function for IGs, our results suggest a possible scenario for the targeting of these genes by parental genomic imprinting during mammalian evolution

Identification d'un réseau de gènes soumis à empreinte génomique parentale et son rôle dans le contrôle des transitions entre prolifération, quiescence et différenciation.

L'empreinte génomique parentale est un mécanisme de régulation épigénétique conduisant à la répression d'un allèle d'un gène en fonction de son origine parentale. Ce mécanisme affecte un nombre restreint de gènes chez les mammifères métathériens et euthériens. Ces gènes, dits gènes soumis à empreinte (GSE), ont des fonctions moléculaires variées et sans lien apparent. Cependant, deux thèmes reviennent de manière récurrente dans leurs fonctions: le contrôle de la croissance embryonnaire et la tumorigenèse. Ma thèse a consisté à démontrer l'existence d'un lien fonctionnel entre les GSE. Nous montrons que les GSE s'inscrivent dans un même réseau de co-expression transcriptionnelle et qu'ils sont co-régulés dans différentes situations biologiques lors des transitions entre les différents états cellulaires. En effet, une induction coordonnée de la plupart des GSE a lieu lors des sorties du cycle cellulaire, réversibles (quiescence) ou non (différenciation). Les perturbations individuelles de l'expression de plusieurs GSE dans le modèle des pré-adipocytes 3T3-L1 confirment un rôle du réseau des GSE dans le contrôle des transitions entre prolifération, quiescence et différenciation. De plus, l'analyse des gènes bi-alléliques inclus dans le même réseau de co-régulation que les GSE montre un enrichissement en gènes de la matrice extracellulaire. La fonction associée à ce réseau serait donc le contrôle des transitions entre les différents états cellulaires, via le remodelage de la matrice extracellulaire. Pour conclure, outre l'identification d'une fonction commune aux GSE, nos résultats suggèrent un scénario pour le ciblage de ces gènes par l'empreinte génomique parentale au cours de l'évolution des mammifères.Genomic imprinting is an epigenetic mechanism leading to the repression of one allele of a gene, depending on its parental origin. This mechanism affects a small number of genes in metatherian and eutherian mammals. These genes, named imprinted genes (IGs), display various molecular functions and thus seem unrelated. However, their alterations are frequently associated with the control of embryonic growth and tumorigenesis. My PhD project has consisted in demonstrating a functional link between IGs. We show that IGs are frequently co-expressed and belong to a common gene network. They are co-regulated in biological situations corresponding to the transitions between different cellular states. Coordinated induction of most IGs takes place at the outputs of the cell cycle. Loss and gain of function experiments of several IGs in the 3T3-L1 pre-adipocyte model demonstrate a role of the IG network in controlling transitions between cellular states (proliferation, quiescence and differentiation). In addition to IGs, this network also includes bi-allelic genes, with many extracellular matrix genes. Therefore, the function associated with the IG network could be the fine control of transitions between cellular states through a remodeling of the extracellular matrix.To conclude, in addition to the identification of a common cellular function for IGs, our results suggest a possible scenario for the targeting of these genes by parental genomic imprinting during mammalian evolution.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Developing a toolbox to study the rabbit methylome and its alteration in IUGR cases during gestation

Epigenome is the essential mediator of the effect of environmental exposures on development. Epigenetic studies show that alterations in DNA methylation marks are associated with developmental reprogramming and linked to environmental exposures. Our objective is to develop different Intra Uterine Growth Restriction (IUGR) rabbit models and to study the alterations occurring in the methylome of the placental unit throughout gestation. The relevance of the rabbit model resides in its placentation similarity with humans. As little is known about rabbit epigenome, we are developing a strategy to establish a toolbox to study the rabbit methylome. First, studying the methylome of the placental unit will be performed by MeDIP-seq on pooled samples to generate a methylation overview in both placenta (fetal compartment) and decidua (maternal compartment) at term. In a second time, a microarray will be designed for individual analysis to establish early epigenetic events related to placenta from IUGR. The array design will integrate methylated sequences from MeDIP-seq data but also promoters of differentially expressed genes obtained by transcriptomic analysis performed on the same samples. Finally, to validate our results, methylated regions of interest will be studied by pyrosequencing after bisulfite treatment. The identification of critical epigenetic marks alterations associated with IUGR will allow a better understanding of the IUGR process and an identification of key actors in placental function. In future studies, these marks could be linked to the establishment of a specific phenotype at adulthood and tested as biomarkers at birth to define the risk of developing an adverse phenotype. In addition, this study will present the first overview of the rabbit methylome and lead to the development of epigenomic tools that can be used systematically

Developing a toolbox to study the rabbit methylome and its alteration in IUGR cases during gestation

International audienceEpigenome is the essential mediator of the effect of environmental exposures on development. Epigenetic studies show that alterations in DNA methylation marks are associated with developmental reprogramming and linked to environmental exposures. Our objective is to develop different Intra Uterine Growth Restriction (IUGR) rabbit models and to study the alterations occurring in the methylome of the placental unit throughout gestation. The relevance of the rabbit model resides in its placentation similarity with humans. As little is known about rabbit epigenome, we are developing a strategy to establish a toolbox to study the rabbit methylome. First, studying the methylome of the placental unit will be performed by MeDIP-seq on pooled samples to generate a methylation overview in both placenta (fetal compartment) and decidua (maternal compartment) at term. In a second time, a microarray will be designed for individual analysis to establish early epigenetic events related to placenta from IUGR. The array design will integrate methylated sequences from MeDIP-seq data but also promoters of differentially expressed genes obtained by transcriptomic analysis performed on the same samples. Finally, to validate our results, methylated regions of interest will be studied by pyrosequencing after bisulfite treatment. The identification of critical epigenetic marks alterations associated with IUGR will allow a better understanding of the IUGR process and an identification of key actors in placental function. In future studies, these marks could be linked to the establishment of a specific phenotype at adulthood and tested as biomarkers at birth to define the risk of developing an adverse phenotype. In addition, this study will present the first overview of the rabbit methylome and lead to the development of epigenomic tools that can be used systematically

Genome-wide analysis in the mouse embryo reveals the importance of DNA methylation for transcription integrity

DNA methyltrasferases play important role during mouse embryo development. Here the authors reveal the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos genome-wide