Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in Saccharomyces cerevisiae

Abstract

Abstract The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.