Combinatorial Genetic Control of Rpd3S Through Histone H3K4 and H3K36 Methylation in Budding Yeast.

Much of euchromatin regulation occurs through reversible methylation of histone H3 lysine-4 and lysine-36 (H3K4me and H3K36me). Using the budding yeast Saccharomyces cerevisiae, we previously found that levels of H3K4me modulated temperature sensitive alleles of the transcriptional elongation complex Spt6-Spn1 through an unknown H3K4me effector pathway. Here we identify the Rpd3S histone deacetylase complex as the H3K4me effector underlying these Spt6-Spn1 genetic interactions. Exploiting these Spt6-Spn1 genetic interactions, we show that H3K4me and H3K36me collaboratively impact Rpd3S function in an opposing manner. H3K36me is deposited by the histone methyltransferase Set2 and is known to promote Rpd3S function at RNA PolII transcribed open reading frames. Using genetic epistasis experiments, we find that mutations perturbing the Set2-H3K36me-Rpd3S pathway suppress the growth defects caused by temperature sensitive alleles of SPT6 and SPN1, illuminating that this pathway antagonizes Spt6-Spn1 Using these sensitive genetic assays, we also identify a role for H3K4me in antagonizing Rpd3S that functions through the Rpd3S subunit Rco1, which is known to bind H3 N-terminal tails in a manner that is prevented by H3K4me. Further genetic experiments reveal that the H3K4 and H3K36 demethylases JHD2 and RPH1 mediate this combinatorial control of Rpd3S. Finally, our studies also show that the Rpd3L complex, which acts at promoter-proximal regions of PolII transcribed genes, counters Rpd3S for genetic modulation of Spt6-Spn1, and that these two Rpd3 complexes balance the activities of each other. Our findings present the first evidence that H3K4me and H3K36me act combinatorially to control Rpd3S.

Lee KY ，Ranger M ，Meneghini MD 《G3-Genes Genomes Genetics》

H3K4 Methylation Dependent and Independent Chromatin Regulation by JHD2 and SET1 in Budding Yeast.

Set1 and Jhd2 regulate the methylation state of histone H3 lysine-4 (H3K4me) through their opposing methyltransferase and demethylase activities in the budding yeast Saccharomyces cerevisiae H3K4me associates with actively transcribed genes and, like both SET1 and JHD2 themselves, is known to regulate gene expression diversely. It remains unclear, however, if Set1 and Jhd2 act solely through H3K4me. Relevantly, Set1 methylates lysine residues in the kinetochore protein Dam1 while genetic studies of the S. pombe SET1 ortholog suggest the existence of non-H3K4 Set1 targets relevant to gene regulation. We interrogated genetic interactions of JHD2 and SET1 with essential genes involved in varied aspects of the transcription cycle. Our findings implicate JHD2 in genetic inhibition of the histone chaperone complexes Spt16-Pob3 (FACT) and Spt6-Spn1 This targeted screen also revealed that JHD2 inhibits the Nrd1-Nab3-Sen1 (NNS) transcription termination complex. We find that while Jhd2's impact on these transcription regulatory complexes likely acts via H3K4me, Set1 governs the roles of FACT and NNS through opposing H3K4-dependent and -independent functions. We also identify diametrically opposing consequences for mutation of H3K4 to alanine or arginine, illuminating that caution must be taken in interpreting histone mutation studies. Unlike FACT and NNS, detailed genetic studies suggest an H3K4me-centric mode of Spt6-Spn1 regulation by JHD2 and SET1 Chromatin immunoprecipitation and transcript quantification experiments show that Jhd2 opposes the positioning of a Spt6-deposited nucleosome near the transcription start site of SER3, a Spt6-Spn1 regulated gene, leading to hyper-induction of SER3 In addition to confirming and extending an emerging role for Jhd2 in the control of nucleosome occupancy near transcription start sites, our findings suggest some of the chromatin regulatory functions of Set1 are independent of H3K4 methylation.

Lee KY ，Chen Z ，Jiang R ，Meneghini MD ... -  《-》

DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

Drouin S ，Laramée L ，Jacques PÉ ，Forest A ，Bergeron M ，Robert F ... -  《PLoS Genetics》

A conserved genetic interaction between Spt6 and Set2 regulates H3K36 methylation.

The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. However, the nature of the requirement for Spt6 has remained elusive. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated several highly clustered, dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. We also show that SET2sup mutations overcome the requirement for certain Set2 domains for H3K36 methylation. In vivo, SET2sup mutants have elevated levels of H3K36 methylation and the purified Set2sup mutant protein has greater enzymatic activityin vitro. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occurs at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple Set2 interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.

Gopalakrishnan R ，Marr SK ，Kingston RE ，Winston F ... -  《-》

Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription.

Carrozza MJ ，Li B ，Florens L ，Suganuma T ，Swanson SK ，Lee KK ，Shia WJ ，Anderson S ，Yates J ，Washburn MP ，Workman JL ... -  《Cell》