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 ... -  《-》

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions.

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

Gopalakrishnan R ，Winston F 《-》

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》

Roles for Ctk1 and Spt6 in regulating the different methylation states of histone H3 lysine 36.

Youdell ML ，Kizer KO ，Kisseleva-Romanova E ，Fuchs SM ，Duro E ，Strahl BD ，Mellor J ... -  《-》

The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae.

Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.

Reim NI ，Chuang J ，Jain D ，Alver BH ，Park PJ ，Winston F ... -  《-》