Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that impact genome regulation and cell physiology throughout their RNA-centered life cycle 1 . Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases 2-4 . Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m 6 A RNA methylation as a novel means of ERV restriction. Methylation of ERV mRNAs is catalyzed by the complex of methyltransferase-like METTL3/METTL14 5 proteins whose depletion, 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'UTR region. Using controlled auxindependent degradation of the METTL3/METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m 6 A catalysis. By monitoring mRNA degradation rates upon METTL3/14 double degron, we further proved that m 6 A methylation destabilizes IAP transcripts. Finally, similarly to m 6 A writers, triple knockout of the m 6 A readers YTHDF1, DF2 and DF3 6 increased IAP mRNA abundance. This study sheds light onto a novel function of RNA methylation in protecting cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.

The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1β), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed withO-linked β-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer ofN-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed thatO-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss ofO-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal ofO-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed thatO-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.