Regulation of transcription elongation and intragenic transcription by the histone chaperone Spt6

Abstract

Precise regulation of transcription is essential for cellular growth and development. Multiple factors are involved in the regulation of transcription by RNA polymerase II (RNAPII). One such factor is the histone chaperone Spt6, which interacts with RNAPII, histones, and other transcription elongation factors during transcription. Mutations in SPT6 cause defects in nucleosome organization, histone H3K36 methylation, and transcription, indicating the requirement for Spt6 in the regulation of both transcription and co-transcriptional processes. spt6 mutants also show a genome-wide increase in transcription initiation from within the gene bodies (hereafter referred to as intragenic transcription). Many of the functions of Spt6 are conserved between yeast and mammalian cells, underscoring its importance in gene regulation. We have taken genetic and biochemical approaches in Saccharomyces cerevisiae to understand the function of Spt6 in regulating intragenic transcription and its role in the transcription elongation complex. In chapter 2 of this dissertation, we describe a genetic selection to identify mutations that repress intragenic transcription in an spt6 mutant. This selection identified dominant mutations in the gene encoding the histone methyltransferase Set2. The SET2 mutations encode a hyperactive enzyme that rescues the H3K36 methylation defect in spt6 mutants. We show that this genetic interaction between SPT6 and SET2 is conserved in the distantly related fission yeast, Schizosaccharomyces pombe, suggesting a conserved role for Spt6 in regulating H3K36 methylation. In chapter 3, we describe using mass spectrometry to characterize the differences in RNAPII-interacting proteins between wild-type and spt6 mutant cells. We show that Spt6 is required for the association of multiple transcription elongation factors with RNAPII, including Spt5 and the PAF complex. We also identify a previously unknown role for Spt6 in the recruitment of the capping enzyme Abd1 to transcribed regions. Taken together, this work provides new insights into the function of Spt6 as a link between transcription elongation and co-transcriptional processes.