Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling

Abstract

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3′-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphateregulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD.H2A.Z | SWRc I n eukaryotic cells, RNA polymerase II (RNAP II) transcribes all protein coding genes as well as a number of noncoding RNA genes, including those for most snRNAs and microRNAs. Expression of RNAP II-transcribed genes is highly regulated, ensuring that the timing of production, quantity, and structure of the RNAs generated is appropriate. Much of this regulation occurs on the genes themselves, whose accessibility is limited by nucleosomes that can form densely packed chromatin (reviewed in refs. 1 and 2). A significant amount of regulation, however, targets RNAP II itself. Specifically, the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAP II, is subject to a multitude of phosphorylation and dephosphorylation events that mark different steps of the RNAP II transcription cycle (recently reviewed in refs. 3-5). Although many of these modifications occur generally, some appear to play critical roles in expression of specific genes (6-8). The CTD consists of a tandemly repeated heptapeptide, whose consensus sequence, Tyr , is conserved in yeast, which harbors 26 repeats, and vertebrates, with 52 repeats. The emerging picture places this unusual structure in a highly important role in coordinating transcription by RNAP II with other events in the generation and maturation of RNAs.Since its discovery, numerous studies have focused on the role of the entire CTD and the function of individual amino acids within its repeats. At promoters, the unphosphorylated CTD interacts directly with subunits of the Mediator complex (9), which is required for Mediator's role in stimulating transcription (10). Additionally, early studies determined that the CTD is required for full activation by some promoter-bound activators (11-13) whereas it was later shown that the CTD can mediate the influence of activators on pre-mRNA processing (14-16).Several genome-wide analyses uncovered a pattern of CTD phosphorylation that occurs once RNAP II clears promoters (e.g., refs. 17 and 18). Phosphory...

Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

“…Striking correlations between 3ʹ-extension index, high pThr4, and low pSer2 levels implicate pThr4 in snoRNA termination. As α-pThr4 antibodies yield modest ChIP signals, we examined pThr4 signals in other genome-wide studies of this mark (15,40). Similar reciprocal patterns of pThr4 and pSer2 profiles were evident in pThr4 ChIP data obtained with a different (6D7) α-pThr4 antibody (40, 41) (SI Appendix, Fig.…”

“…S4C). Further, a genetic link between Thr4 and chromatin remodeling through the histone variant Htz1 has been reported (15). Consistent with a termination defect, Htz1, a histone H2A variant that yields more labile nucleosomes (68,69), was highly enriched downstream of snoRNAs that display read-through in T4A (70) (SI Appendix, Fig.…”

“…The CID domain of Rtt103 (Rtt103-CID) used for NMR titration and chemical shift assignments was prepared as described previously (20) and 15 N or 15 N, 13 C labeled. The diheptad peptides were purchased from AnaSpec or VCPBio Lab.…”

“…NMR data were processed with NMRPipe (75) and analyzed within CcpNmr (76). The complex of Rtt103-CID with diheptad peptides was formed by titrating the peptide into the CID and monitoring the change of 15 N-edited HSQC spectra until saturation was observed. The chemical shifts of backbone and side-chain resonances were assigned using standard triple resonance NMR experiments including 3D HNCO, HNCACB, and CBCA(CO)NH (77).…”

“…The mechanistic consequences of phosphorylating Ser5 and Ser2 have been well documented (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). However, the role of Thr4 phosphorylation (pThr4), and even the necessity of Thr4 for cellular survival, appears to differ among closely related species and between growth conditions within a given species (12)(13)(14)(15). Recent mass spectrometric analysis of an extensively engineered CTD revealed a paucity of pThr4, raising questions about the importance of this mark (16).…”