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Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout their RNA-centred life cycle.sup.1. Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases.sup.2,3. Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m.sup.6A RNA methylation as a way to restrict ERVs. Methylation of ERV mRNAs is catalysed by the complex of methyltransferase-like METTL3-METTL14.sup.4 proteins, and we found that depletion of METTL3-METTL14, along with their accessory subunits WTAP and ZC3H13, led to increased mRNA abundance of intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5' untranslated region. Using controlled auxin-dependent degradation of the METTL3-METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m.sup.6A catalysis. By monitoring chromatin states and mRNA stability upon METTL3-METTL14 double depletion, we found that m.sup.6A methylation mainly acts by reducing the half-life of IAP mRNA, and this occurs by the recruitment of the YTHDF family of m.sup.6A reader proteins.sup.5. Together, our results indicate that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent. A CRISPR screen in mouse embryonic stem cells shows that transcripts derived from endogenous retroviruses are destabilized by m.sup.6A RNA methylation., Author(s): Tomasz Chelmicki [sup.1] , Emeline Roger [sup.1] , Aurélie Teissandier [sup.1] , Mathilde Dura [sup.1] , Lorraine Bonneville [sup.1] , Sofia Rucli [sup.1] , François Dossin [sup.2] , Camille [...]

Mouse embryonic stem cells have an inherent propensity to explore gene regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). Whereas before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes independent of ERK. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3 links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation. [ABSTRACT FROM AUTHOR]

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 con trast, three mutants (Dnmt3I, 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.

Gnas is a complex gene with multiple imprinted promoters. The upstream Nesp and Nespas/Gnasxl promoters are paternally and maternally methylated, respectively. The downstream promoter for the stimulatory G protein [alpha]-subunit ([G.sub.s][alpha]) is unmethylated, although in some tissues (e.g., renal proximal tubules), [G.sub.s][alpha] is poorly expressed from the paternal allele. Just upstream of the [G.sub.s][alpha] promoter is a primary imprint mark (1A region) where maternal-specific methylation is established during oogenesis. Pseudohypoparathyroidism type 1B, a disorder of renal parathyroid hormone resistance, is associated with loss of 1A methylation. Analysis of embryos of Dnmt3[L.sup.-/-] mothers (which cannot methylate maternal imprint marks) showed that Nesp, Nespas/Gnasxl, and 1A imprinting depend on one or more maternal primary imprint marks. We generated mice with deletion of the 1A differentially methylated region. These mice had normal Nesp-Nespas/Gnasxl imprinting, indicating that the Gnas locus contains two independent imprinting domains (Nespas-Nespas/Gnasxl and 1A-[G.sub.s][alpha]) controlled by distinct maternal primary imprint marks. Paternal, but not maternal, 1A deletion resulted in [G.sub.s][alpha] overexpression in proximal tubules and evidence for increased parathyroid hormone sensitivity but had no effect on [G.sub.s][alpha] expression in other tissues where [G.sub.s][alpha] is normally not imprinted. The 1A region is a maternal imprint mark that contains one or more methylation-sensitive cis-acting elements that suppress [G.sub.s][alpha] expression from the paternal allele in a tissue-specific manner. genomic imprinting | pseudohypoparathyroidism | DNA methylation | guanine nucleotide binding protein

DNA methylation is a major epigenetic marker for differentiation, genome integrity and parental imprinting. Technological advances now allow us to decipher germline and embryonic methylomes, improving our understanding of epigenetic regulation during gametogenesis and embryogenesis. A new era has also been opened by the development of genetic engineering techniques in molecular biology, now amenable to epigenome editing. [ABSTRACT FROM AUTHOR]

Complementary sets of genes are epigenetically silenced in male and female gametes in a process termed genomic imprinting. The Dnmt3L gene is expressed during gametogenesis at stages where genomic imprints are established. Targeted disruption of Dnmt3L caused azoospermia in homozygous males, and heterozygous progeny of homozygous females died before midgestation. Bisulfite genomic sequencing of DNA from oocytes and embryos showed that removal of Dnmt3L prevented methylation of sequences that are normally maternally methylated. The defect was specific to imprinted regions, and global genome methylation levels were not affected. Lack of maternal methylation imprints in heterozygous embryos derived from homozygous mutant oocytes caused biallelic expression of genes that are normally expressed only from the allele of paternal origin. The key catalytic motifs characteristic of DNA cytosine methyltransferases have been lost from Dnmt3L, and the protein is more likely to act as a regulator of imprint establishment than as a DNA methyltransferase., Genomic imprinting imposes a requirement for biparental reproduction. Uniparental mammalian conceptuses are inviable (1-3), and lack of imprinting of specific chromosomes or chromosome segments as a result of uniparental isodisomy [...]

It has been established that the ultimate cause of the recessive autosomal disorder known as ICF syndrome is defective DNA methylation. It was found that five unrelated ICF patients had mutations in both alleles of the gene that encodes DNA methyltransferase 3B. Cytosine methylation is vital for the organization and stabilization of a certain type of heterochromatin, and this methylation seems to be undertaken by an enzyme specialized for the role. It is possible that methylation is needed for the assembly of the silencing foci around condensation of satellite DNA, for the recruitment of silencing proteins to the foci or for the migration of silent genes into the foci.

DNA methylation patterns in preimplantation mouse development have been studied by analysis of monoclonal antibody-to-5-methylcytosine (5-MeC) binding on metaphase chromosomes. At each cell stage certain chromosome patterns appeared. It appears that chromosome demethylation is associated with each DNA replication. A passive mechanism appears to be predominant in early development.

Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout their RNA-centred life cycle1. Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases2,3. Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m6A RNA methylation as a way to restrict ERVs. Methylation of ERV mRNAs is catalysed by the complex of methyltransferase-like METTL3–METTL144 proteins, and we found that depletion of METTL3–METTL14, along with their accessory subunits WTAP and ZC3H13, led to increased mRNA abundance of intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5′ untranslated region. Using controlled auxin-dependent degradation of the METTL3–METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m6A catalysis. By monitoring chromatin states and mRNA stability upon METTL3–METTL14 double depletion, we found that m6A methylation mainly acts by reducing the half-life of IAP mRNA, and this occurs by the recruitment of the YTHDF family of m6A reader proteins5. Together, our results indicate that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.A CRISPR screen in mouse embryonic stem cells shows that transcripts derived from endogenous retroviruses are destabilized by m6A RNA methylation. [ABSTRACT FROM AUTHOR]