COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.

Summary Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions.

The TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish due to challenges of distinguishing global versus locus-specific effects. Here we show that TET1/2/3 triple knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding gene expression decrease in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taken together with further genome-wide methylation and TET1 and DNMT3B ChIP-Seq analysis, we conclude that the TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the ⌬ubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation in vivo.Transcription is a highly coordinated and orchestrated process. It takes place on the DNA template that is packaged into chromatin in the nucleus. The chromatin is an array of nucleosomes in which 146 base pairs of DNA (in each nucleosome) are wrapped around the histone octamer (27). Each octamer consists of two molecules each of the histones H2A, H2B, H3, and H4 (27). Nucleosomal arrays fold into a 30-nm fiber upon incorporation of the linker histone H1 (19,22).Histones are subject to posttranslational modifications such as acetylation, phosphorylation, ubiquitination, and methylation, and these modifications are known to play important roles in epigenetic regulation of transcription (33,44). Most modifications were originally observed on the N-terminal tails of histones, with the exception of ubiquitination, which occurs on the C-terminal tails of H2A and H2B (13). However, several novel posttranslational modifications have been identified recently in the core region of the histones (45). Histone modifications alter DNA-histone interactions within and between nucleosomes, and thus they affect the higher-order chromatin structure. Alternatively, combinations of histone modifications present an interaction surface for other proteins that translate the so-called "histone code" into a gene expression pattern (42), explaining how the same chemical modification can have different functional consequences depending on the target amino acid residue.Methylation of histone H3 at lysine 4 (K4) is associated with active chromatin in a wide range of eukaryotic organis...

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five JNK/JUN family genes as key barriers of DE differentiation. The JNK/JUN pathway does not act through directly inhibiting the DE enhancers. Instead JUN co-occupies ESC enhancers with OCT4, NANOG and SMAD2/3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2/3 chromatin binding from ESC to DE enhancers. Therefore, the JNK/JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.

Rad26p, a yeast homologue of human Cockayne syndrome B with an ATPase activity, plays a pivotal role in stimulating DNA repair at the coding sequences of active genes. On the other hand, DNA repair at inactive genes or silent areas of the genome is not regulated by Rad26p. However, how Rad26p recognizes DNA lesions at the actively transcribing genes to facilitate DNA repair is not clearly understood in vivo. Here, we show that Rad26p associates with the coding sequences of genes in a transcription-dependent manner, but independently of DNA lesions induced by 4-nitroquinoline-1-oxide in Saccharomyces cerevisiae. Further, histone H3 lysine 36 methylation that occurs at the active coding sequence stimulates the recruitment of Rad26p. Intriguingly, we find that Rad26p is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner. However, Rad26p does not recognize DNA lesions in the absence of active transcription. Together, these results provide an important insight as to how Rad26p is delivered to the damage sites at the active, but not inactive, genes to stimulate repair in vivo, shedding much light on the early steps of transcription-coupled repair in living eukaryotic cells.

In the above article, the following grant funding should have been included in the Acknowledgments section: M.G. is funded by NIH grants R37 DK44746 and RO1 HL65440. M.B. is funded by NIH grant R01 DK070687.

Although Sgf73p, a yeast homologue of human Sca7p, has recently been implicated as a new component of Spt-Ada-Gcn5-acetyltransferase (SAGA), its association with SAGA and functional role in regulation of transcription remain unknown in vivo. Here, using a chromatin immunoprecipitation (ChIP) assay, we show in vivo that, like SAGA, Sgf73p is recruited to the upstream activating sequence (UAS) of a SAGA-dependent gene, GAL1, in an activator-dependent manner. Further, Sgf73p is required for recruitment of SAGA to the GAL1 UAS, and facilitates formation of the preinitiation complex (PIC) assembly at the GAL1 promoter. When PIC is not formed in Δsgf73, histone H3 is not evicted from the GAL1 promoter. Interestingly, PIC formation at GAL1 is not regulated by histone H3 acetylation or histone acetyltransferase (HAT) activity of SAGA. Similarly, Sgf73p facilitates PIC formation at another SAGA-dependent gene, ADH1, independent of histone H3 acetylation or HAT. In contrast, Sgf73p stimulates PIC formation at PHO84 (a SAGA-dependent gene), in a HAT-dependent-manner. Collectively, our data reveal that Sgf73p is required for SAGA recruitment, and stimulates PIC formation either in a HAT-dependent or -independent manner, providing significant information on how Sgf73p and possibly human Sca7p function physiologically.