The TAF9 C-Terminal Conserved Region Domain Is Required for SAGA and TFIID Promoter Occupancy To Promote Transcriptional Activation

Abstract

A common function of the TFIID and SAGA complexes, which are recruited by transcriptional activators, is to deliver TBP to promoters to stimulate transcription. Neither the relative contributions of the five shared TBP-associated factor (TAF) subunits in TFIID and SAGA nor the requirement for different domains in shared TAFs for transcriptional activation is well understood. In this study, we uncovered the essential requirement for the highly conserved C-terminal region (CRD) of Taf9, a shared TAF, for transcriptional activation in yeast. Transcriptome profiling performed under Gcn4-activating conditions showed that the Taf9 CRD is required for induced expression of ϳ9% of the yeast genome. The CRD was not essential for the Taf9-Taf6 interaction, TFIID or SAGA integrity, or Gcn4 interaction with SAGA in cell extracts. Microarray profiling of a SAGA mutant (spt20⌬) yielded a common set of genes induced by Spt20 and the Taf9 CRD. Chromatin immunoprecipitation (ChIP) assays showed that, although the Taf9 CRD mutation did not impair Gcn4 occupancy, the occupancies of TFIID, SAGA, and the preinitiation complex were severely impaired at several promoters. These results suggest a crucial role for the Taf9 CRD in genome-wide transcription and highlight the importance of conserved domains, other than histone fold domains, as a common determinant for TFIID and SAGA functions.

Mutational analysis of TAF6 revealed the essential requirement of the histone‐fold domain and the HEAT repeat domain for transcriptional activation

“…For the ChIP analysis, we selected the UAS regions of ARG1, TRP3, BNA1, MET28, GGC1 and YLR152C because their expression was up-regulated upon amino acid starvation imposed by SM [34] and they were also shown to be bound by Gcn4 in cells cultured in SM-containing medium [34,47]. For the ChIP analysis, we selected the UAS regions of ARG1, TRP3, BNA1, MET28, GGC1 and YLR152C because their expression was up-regulated upon amino acid starvation imposed by SM [34] and they were also shown to be bound by Gcn4 in cells cultured in SM-containing medium [34,47].…”

“…Gcn4 actively recruits several co-activators, GTFs and RNA pol II to several target promoters including that of ARG1 promoter [34,[48][49][50]. Gcn4 actively recruits several co-activators, GTFs and RNA pol II to several target promoters including that of ARG1 promoter [34,[48][49][50].…”

“…The PolIII transcribed scR1 RNA was used as the endogenous control as described previously [34]. Extracted RNA was ethanol precipitated with sodium acetate and pelleted by centrifugation.…”

“…For immunoprecipitation reactions, Protein G-Sepharose beads (GE Healthcare) were washed with 19 PBS. The fold enrichment was calculated by the 2 ÀDDCT relative-quantitation method [64,65] and as described previously [34]. For the TAP tagged ChIP assay, IgG-Sepharose beads (GE Healthcare) were blocked with 1 mgÁmL À1 BSA and incubated with chromatin extracts for 2 h at 4°C.…”

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

“…S8A-D, upper panels), and identified 223 genes with increased Rpb3 occupancies averaged over the CDSs of greater than or equal to twofold on Gcn4 induction (Supplemental Fig. S9, above dashed diagonal; Supplemental File S1, "211 genes"), of which ∼80% were found by microarray expression analysis (Saint et al 2014) to exhibit a comparable increase in mRNA abundance on SM treatment (P = 1 × 10…”