RESUMEN
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.
Asunto(s)
Retrovirus Endógenos/genética , Genes de Partícula A Intracisternal/genética , Metilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Técnicas de Inactivación de Genes , Semivida , Metiltransferasas/metabolismo , Ratones , Células Madre Embrionarias de Ratones , Proteínas Nucleares/metabolismo , Factores de Empalme de ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/química , Proteínas de Unión al ARN/metabolismoRESUMEN
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 TIF1ß), 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 with O-linked ß-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 of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-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 of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-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 that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.