Summary Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist -RNA binding proteins have recently been identified, including SPEN 1 – 3 , the loss of which has been associated with deficient XCI at multiple loci 2 – 6 . Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly dampens expression of genes that escape XCI. We show that SPEN is immediately recruited to the X-chromosome upon Xist up-regulation, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC domain as a major effector of SPEN’s gene silencing function, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.

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.

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Histone acetyl transferases (HATs) play distinct roles in many cellular processes and are frequently misregulated in cancers. Here, we study the regulatory potential of MYST1-(MOF)-containing MSL and NSL complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. We find that both complexes influence transcription by targeting promoters and TSS-distal enhancers. In contrast to flies, the MSL complex is not exclusively enriched on the X chromosome, yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix, the major repressor of Xist lncRNA. MSL depletion leads to decreased Tsix expression, reduced REX1 recruitment, and consequently, enhanced accumulation of Xist and variable numbers of inactivated X chromosomes during early differentiation. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs.DOI: http://dx.doi.org/10.7554/eLife.02024.001

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Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.

27 28 29 Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated 30 retroviral sequences that impact genome regulation and cell physiology throughout 31 their RNA-centered life cycle 1 . Failure to repress ERVs is associated with cancer, 32 infertility, senescence and neurodegenerative diseases 2-4 . Here, using an unbiased 33 genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify 34 m 6 A RNA methylation as a novel means of ERV restriction. Methylation of ERV mRNAs 35 is catalyzed by the complex of methyltransferase-like METTL3/METTL14 5 proteins 36 whose depletion, along with their accessory subunits, WTAP and ZC3H13, led to 37 increased mRNA abundance of Intracisternal A-particles (IAPs) and related ERVK 38 elements specifically, by targeting their 5'UTR region. Using controlled auxin-39 dependent degradation of the METTL3/METTL14 enzymatic complex, we showed that 40 IAP mRNA and protein abundance is dynamically and inversely correlated with m 6 A 41 catalysis. By monitoring mRNA degradation rates upon METTL3/14 double degron, we 42 further proved that m 6 A methylation destabilizes IAP transcripts. Finally, similarly to 43 m 6 A writers, triple knockout of the m 6 A readers YTHDF1, DF2 and DF3 6 increased IAP 44 mRNA abundance. This study sheds light onto a novel function of RNA methylation in 45 protecting cellular integrity by clearing reactive ERV-derived RNA species, which may 46 be especially important when transcriptional silencing is less stringent.repressors, by transducing cells with single guide (sg)RNAs against the KRAB-associated 80 protein 1 (KAP1) 11 (Extended Data Fig. 1d,e, Supplementary Fig. 1, Supplementary Table 2, 81 Supplementary Table 3). 4For the screen, we transduced IAPEz-reporter cells with a lentiviral genome-wide 83 sgRNA library at multiplicity of infection (MOI)= 0.2-0.3 12 (Fig. 1b). Frequencies of sgRNAs 84 upon blasticidin selection (5, 7 and 9 days) versus non-selected conditions were assessed 85 via deep sequencing and candidate genes were identified using Model-based Analysis of 86 Genome-wide CRISPR/Cas9 Knockout (MAGeCK) 13 . Efficiency of selection was evidenced 87 by drop-out of control intergenic sgRNAs and genes were ranked based on sgRNA P-values 88 ( Fig. 1c, Supplementary Table 4, Supplementary Table 5). Although genome-wide screens of 89 this magnitude typically suffer from low-statistical confidence, we identified several-but not 90 all-genes previously associated with ERV repression (Resf1, Trp53, Daxx, Atrx, Uhrf1, 91 Cbx1, Dnmt1) 14-17 among the top 100 hits. Obtaining an incomplete list of known regulators 92 is a common outcome of genome-wide screens which can be due to multiple factors 93 including time-dependent dropout of essential genes with strong effects on cell viability (such 94 as Kap1) 18 , heterogeneity in sgRNA efficiencies or limited representation of sgRNAs. 95Nonetheless, we were able to identify several novel candidates for IAP control 96( Supplementary Table 4), offering a foundation for futu...

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