Coordinate regulation of DNA methyltransferase expression during oogenesis

<p>Abstract</p> <p>Background</p> <p>Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of <it>Dnmt3a </it>and <it>Dnmt3b</it>, as well as a regulator of DNA methylation, <it>Dnmt3L</it>, in isolated female germ cells.</p> <p>Results</p> <p>Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated <it>Snrpn</it>, <it>Peg3 </it>and <it>Igf2r </it>DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the <it>de novo </it>methyltransferase <it>Dnmt3b</it>, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.</p> <p>Conclusion</p> <p>Together these results provide a better understanding of the developmental regulation of <it>Dnmt3a</it>, <it>Dnmt3b </it>and <it>Dnmt3L </it>at the time of <it>de novo </it>methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.</p

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

<p>Abstract</p> <p>Background</p> <p>Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development.</p> <p>Results</p> <p>A developmental study covering the first 12 days following birth was conducted on a <it>Dnmt3L </it>mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in <it>Dnmt3L</it>^-/-males, pointing to a mitotic defect. A temporal expression study showed that expression of <it>Dnmt3L </it>is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in <it>Dnmt3L</it>^+/+germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content.</p> <p>Conclusion</p> <p>Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed.</p

Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI

MORC1 represses transposable elements in the mouse male germline

The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA-methylated and silent genes. ​MORC1-deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that ​MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homologue ​DNMT3L. ​Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify ​MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation

The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution

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

Caractérisation génomique et fonctionnelle de l'empreinte parentale par le modèle murin mutant pour Dnmt3L

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

IDENTIFICATION DU GENE ET DE L'ORIGINE EMBRYONNAIRE DU DEFAUT DE METHYLATION DE L'AON DES PATIENST ICF

LA METHYLATION DE L'ADN DES MAMMIFERES EST IMPLIQUEE DANS DES PROCESSUS AUSSI FONDAMENTAUX QUE LE CONTROLE DES ELEMENTS PARASITES ENDOGENES ET LA REPRESSION GENIQUE DANS LE CONTEXTE PARTICULIER DE L'EMPREINTE ET DE L'INACTIVATION DU CHROMOSOME X. CE TRAVAIL DE THESE S'EST ORGANISE AUTOUR DE DEUX AXES, CONVERGEANT VERS LA COMPREHENSION DE L'IMPORTANCE DE CE PROCESSUS ET DES MECANISMES SOUS-JACENTS AUX TRANSITIONS DE METHYLATION : UNE ETUDE DANS UN CONTEXTE PATHOLOGIQUE, CELUI DU SYNDROME ICF, ET UNE ANALYSE PLUS FONDAMENTALE DES VARIATIONS DE METHYLATION AU COURS DU DEVELOPPEMENT DE DIFFERENTES ESPECES MAMMIFERES. LE SYNDROME ICF EST UNE MALADIE GENETIQUE RARE, ASSOCIANT UNE IMMUNODEFICIENCE, UNE INSTABILITE CHROMOSOMIQUE ET DES ANOMALIES FACIALES, A UN DEFAUT CONSTITUTIF DE METHYLATION. NOUS AVONS ICI REVELE QUE LES SEQUENCES REPETEES OU HETEROCHROMATIQUES ETAIENT LA CIBLE PREFERENTIELLE DE L'HYPOMETHYLATION, ET QUE LES PRINCIPALES CARACTERISTIQUES DE L'INACTIVATION DU CHROMOSOME X N'ETAIENT PAS PERTURBEES PAR LE DEFAUT. CETTE CARACTERISATION A ETE SUIVIE DE L'IDENTIFICATION DU GENE RESPONSABLE DU SYNDROME, CODANT POUR UNE ENZYME IMPLIQUEE DANS L'ETABLISSEMENT DES PROFILS DE METHYLATION AU COURS DU DEVELOPPEMENT, L'ADN-METHYLTRANSFERASE 3B (DNMT3B). LE DEVELOPPEMENT EMBRYONNAIRE PRECOCE ET LA GAMETOGENESE SONT EN EFFET DES PHASES CRITIQUES POUR LA CONSTITUTION DES PROFILS DE METHYLATION DE L'INDIVIDU. NOUS AVONS AINSI CARACTERISE LES CHANGEMENTS QUI S'OPERENT PENDANT CES PERIODES CHEZ LA SOURIS, REVELANT UNE EVOLUTION DIFFERENTE POUR L'EUCHROMATINE ET L'HETEROCHROMATINE, AVEC EN PARTICULIER UNE DYNAMIQUE INATTENDUE DES PROFILS DE METHYLATION DE L'HETEROCHROMATINE NORMALEMENT CONSIDEREE COMME DE L'ADN INERTE. ENFIN, L'IMPORTANCE DE PROFILS DE METHYLATION ADEQUATS A ETE ILLUSTREE PAR L'ETUDE DE CELLULES ES DEFICIENTES EN METHYLATION ET ENFIN, D'EMBRYONS BOVINS ISSUS DE CLONAGE SOMATIQUE, POUR LESQUELS LA REPROGRAMMATION DES PROFILS DE METHYLATION APPARAIT INCOMPLETE.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

Background: Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development. Results: A developmental study covering the first 12 days following birth was conducted on a Dnmt3L mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in Dnmt3L-/- males, pointing to a mitotic defect. A temporal expression study showed that expression of Dnmt3L is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in Dnmt3L+/+ germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content. Conclusion: Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed

Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth

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