Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex

Abstract

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.

Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation

“…Conventional ChIP experiments were conducted as described previously (Qiu et al 2012) using anti-H3 (abcam, ab1791) and anti-Gcn4 (Zhang et al 2008) antibodies and primers summarized in Supplemental Figure S1. For ChIP-seq analysis, DNA libraries for Illumina paired-end sequencing of chromatin immunoprecipitates (PESCI) were prepared as described previously (Cole et al 2014) with modifications indicated in the Supplemental Material.…”

The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

“…With respect to members of Paf1C, loss of the Rtf1, Cdc73, or Leo1 subunits reduces the occupancy of Paf1C on chromatin (18,39,40). In vitro, recombinant Cdc73, Rtf1, and Ctr9 can bind to peptides corresponding to the RNA Pol II CTD phosphorylated on serines 2 and 5 and to peptides corresponding to the phosphorylated Spt5 CTR (35). For Cdc73, this peptide binding activity maps to a domain that adopts a Ras-like fold and is important for Paf1C recruitment in vivo (35,41).…”

“…Previous studies have implicated several proteins in the recruitment of yeast Paf1C to active chromatin, including the transcription elongation factors Spt16-Pob3/FACT, the Ccr4-Not complex, and Spt4-Spt5/DSIF (30)(31)(32). The Bur1-Bur2 protein kinase stimulates the recruitment of Paf1C to RNA Pol II through the phosphorylation of the C-terminal repeats (CTRs) of Spt5 and through a pathway independent of this function (30,(33)(34)(35)(36)(37). In addition, the Kin28 protein kinase promotes the recruitment of Paf1C through phosphorylation of the CTD of RNA Pol II and by facilitating the chromatin association of Bur1-Bur2 (35,38).…”

“…The Bur1-Bur2 protein kinase stimulates the recruitment of Paf1C to RNA Pol II through the phosphorylation of the C-terminal repeats (CTRs) of Spt5 and through a pathway independent of this function (30,(33)(34)(35)(36)(37). In addition, the Kin28 protein kinase promotes the recruitment of Paf1C through phosphorylation of the CTD of RNA Pol II and by facilitating the chromatin association of Bur1-Bur2 (35,38). With respect to members of Paf1C, loss of the Rtf1, Cdc73, or Leo1 subunits reduces the occupancy of Paf1C on chromatin (18,39,40).…”

Vps Factors Are Required for Efficient Transcription Elongation in Budding Yeast

“…Importantly, as observed for conventional elongation factors, the association of Vps proteins with ARG1 or GAL1 CDS was strongly dependent on target gene transcription, occurring at high levels only under conditions where the relevant transcriptional activators are induced (Gcn4) or functional (Gal4). In addition, the TATA promoter element is required for high-level Vps15 and Vps34 occupancies at ARG1 under inducing conditions, and Vps15 and Vps34 occupancies of ARG1 CDS were stimulated by the Pol II CTD kinase Cdk7/ Kin28-a characteristic of numerous factors involved in cotranscriptional histone modifications, mRNA processing or nuclear export, and the elongation or termination phases of transcription (Phatnani and Greenleaf 2006;Govind et al 2007;Pascual-Garcia et al 2008;Ginsburg et al 2009;Govind et al 2010;Qiu et al 2012).…”