Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genes

Abstract

ABSTRACTPaf1C 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 recruitment to Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 remain to be elucidated. Using an in vivo site-specific protein cross-linking strategy, we identified direct interactions between Cdc73 and two components of the elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Through in vitro protein binding assays and crosslinking/mass spectrometry, we show that Cdc73 and Spt6 can interact in the absence of additional factors and propose a binding interface. Rapid depletion of Spt6 dissociated Paf1 from chromatin and altered patterns of Paf1C-dependent histone modifications genome-wide. These results reveal previously unrecognized interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to Paf1C recruitment to active genes in Saccharomyces cerevisiae.