We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.chip-Seq | chromatin modifications | muscle development | transcriptional regulation R egulation of the transcriptome through dynamic changes in chromatin plays an important role in lineage commitment and differentiation. Multiple histone modifications control gene expression through recruitment of factors that alter compaction of the chromatin fiber. Transient and long-term gene silencing is enforced through trimethylation of histone H3 on lysines 9 and 27 (hereafter H3K9me3 and H3K27me3) as well as H4K20, whereas gene activation is regulated by methylation of H3K4 and acetylation of the amino-terminal tails of H3 and H4 (reviewed in refs. 1 and 2). Chromatin modifications are often asymmetrically deposited with respect to the transcription start sites (TSS) of genes. Whereas H3K27me3 is found at promoters, throughout gene bodies, and in intergenic regions, histone tail acetylation and H3K4me3 are predominantly found at promoters and the 5′ ends of genes. On the other hand, H3K36 trimethylation marks gene bodies, signifying the passage of RNA polymerase II (PolII) on actively transcribed genes. Promoter acetylation and H3K4 trimethylation are often coordinated, whereas H3K27 and H3K4 trimethylation are largely anticorrelated, except within bivalent regions poised to adopt either active or repressed states at the appropriate developmental stage (3).Previous studies have shown that the pluripotent state of embryonic stem (ES) cells is in part governed by bivalent nucleosomes, characterized by simultaneous H3K4 and H3K27 trimethylation of nucleosomes in lineage commitment genes (3, 4). During ES cell differentiation, ...

Summary Mono-methylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we reveal novel roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes mono-methylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Mono-methylation mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human ES cells. On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 mono-methylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.

To identify the compendium of distal regulatory elements that govern myogenic differentiation, we generated chromatin state maps based on histone modifications and recruitment of factors that typify enhancers in myoblasts and myotubes. We found a striking concordance between the locations of these newly defined enhancers, MyoD1-binding events, and noncoding RNA transcripts. These enhancers recruit several sequencespecific transcription factors in a spatially constrained manner around MyoD1-binding sites. Remarkably, MyoD1-null myoblasts show a wholesale loss of recruitment of these factors as well as diminished monomethylation of H3K4 (H3K4me1) and acetylation of H3K27 (H3K27ac) and reduced recruitment of Set7, an H3K4 monomethylase. Surprisingly, we found that H3K4me1, but not H3K27ac, could be restored by re-expression of MyoD1 in MyoD1 -/-myoblasts, although re-expression of this factor in MyoD1-null myotubes restored both histone modifications. Our studies identified a role for MyoD1 in condition-specific enhancer assembly through recruitment of transcription factors and histone-modifying enzymes that shape muscle differentiation.

SummaryAutophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes via Foxk-Sin3-mediated transcriptional control.

SUMMARY Low-grade astrocytomas (LGA) carry neomorphic mutations in Isocitrate Dehydrogenase (IDH), concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA and ATRX shRNA in human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo and led to a DNA methylation and transcriptional profile resembling IDH1-mutant human LGAs. The differentiation block was caused by transcriptional silencing of transcription factor SOX2, secondary to disassociation of its promoter from a putative enhancer. This occurred due to reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH-mutant LGA formation implicates impaired NSC differentiation due to repression of SOX2 as an early driver of gliomagenesis.