Spt6 Is Required for the Fidelity of Promoter Selection.

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Doris SM ，Chuang J ，Viktorovskaya O ，Murawska M ，Spatt D ，Churchman LS ，Winston F ... -  《-》

The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations.

H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here we provide genomic and biochemical evidence that the RNA polymerase II (RNA Pol II) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nucleosome reassembly coupled to pervasive incorporation of H2A.Z by mislocalized SWR-C alters chromatin composition and contributes to cryptic initiation. Therefore, chaperone-mediated H2A.Z confinement is crucial for restricting the chromatin signature of gene promoters that otherwise may license or promote cryptic transcription.

Jeronimo C ，Watanabe S ，Kaplan CD ，Peterson CL ，Robert F ... -  《-》

Transcription elongation factors repress transcription initiation from cryptic sites.

Kaplan CD ，Laprade L ，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 《-》

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 ... -  《-》