The Yeast Protein Complex Containing Cdc68 and Pob3 Mediates Core-Promoter Repression Through the Cdc68 N-Terminal Domain

Abstract

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

“…No effect on the majority of tRNA genes is consistent with an earlier study reporting that amino-terminal domain of Spt16 is reduntant for its transcription activation function but necessary for maintaining the chromatin repression of transcription on Pol II-transcribed genes ( Evans et al 1998 ). Yeast tRNA genes reside in the nucleosome-free regions (NFRs) flanked by the positioned nucleosomes on both sides ( Kumar and Bhargava 2013 ).…”

“…4 D). This loss could be due to the reported instability of the mutant Spt16 protein ( Evans et al 1998 ), which results in its lower levels in the spt16-197 cells (Supplemental Fig. S4B,C).…”

FACT and the reorganized nucleosome

“…68 This domain is highly conserved among eukaryotes but deletion of this region causes few phenotypes, including mild sensitivity to HU. 27,45,78,79 As expected from sequence alignments reported earlier, 80 the Spt16 NTD has the overall ''pita bread'' fold of bacterial aminopeptidases, but lacks active site residues and does not have detectable peptidase activity. 27,68 Two conserved grooves in the surface have been identified (Fig.…”

“…43,44 FACT defects could therefore cause the Spt À phenotype directly by failing to perform an initiation function adequately, or indirectly by causing the formation of insufficiently repressive chromatin. 45…”

Spt16 and Pob3 of Saccharomyces cerevisiae Form an Essential, Abundant Heterodimer That Is Nuclear, Chromatin-Associated, and Copurifies with DNA Polymerase α

“…POB3 ( P olymerase o ne- b inding) shares significant sequence similarity with an HMG1-like protein found in a variety of organisms, including human SSRP1 (). SPT16 and POB3 are each essential for viability ( , ); and both Spt16 and Pob3 are highly conserved among a broad range of eukaryotes ( , ).…”