The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (eRNA) regulates transcription of the adjacent MyoD gene, whereas eRNA affects expression of Myogenin in trans. We found thateRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. eRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressingeRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or eRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus,eRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.

Polycomb group (PcG) proteins initiate the formation of repressed chromatin domains and regulate developmental gene expression. A mammalian PcG protein, enhancer of zeste homolog 2 (Ezh2), triggers transcriptional repression by catalyzing the addition of methyl groups onto lysine 27 of histone H3 (H3K27me2/3). This action facilitates the binding of other PcG proteins to chromatin for purposes of transcriptional silencing. Interestingly, there exists a paralog of Ezh2, termed Ezh1, whose primary function remains unclear. Here, we provide evidence for genome-wide association of Ezh1 complex with active epigenetic mark (H3K4me3), RNA polymerase II (Pol II), and mRNA production. Ezh1 depletion reduced global Pol II occupancy within gene bodies and resulted in delayed transcriptional activation during differentiation of skeletal muscle cells. Conversely, overexpression of wild-type Ezh1 led to premature gene activation and rescued Pol II occupancy defects in Ezh1-depleted cells. Collectively, these findings reveal a role for a PcG complex in promoting mRNA transcription.

SUMMARY Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart due to the presence of a ~25kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation.

SUMMARY Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation and mRNA processing. Here, we report that depleting Spt6 in ESCs reduced expression of pluripotency factors, increased expression of cell lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective down-regulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers where Spt6 controls histone H3K27 acetylation and methylation, and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as a histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.