We study the regulation of amino acid biosynthetic genes in budding yeast as a means of dissecting mechanisms of transcriptional control of gene expression in vivo. Transcription of these genes is coordinately induced by transcriptional activator Gcn4 during amino acid limitation, in a response known as general amino acid control (GAAC). Gcn4 expression is coupled to amino acid levels through a translational control mechanism that mediates increased synthesis of Gcn4 under starvation conditions. We showed previously that efficient transcriptional activation by Gcn4 depends on its recruitment of coactivator complexes Mediator, SAGA, SWI/SNF, and RSC, which enhance in turn the recruitment of general transcription factors and RNA Polymerase II (Pol II) to the promoter to stimulate preinitiation complex (PIC) assembly. We further demonstrated that SAGA is recruited co-transcriptionally to coding sequences by association with the Ser5-phosphorylated C-terminal domain (CTD) of Pol II (Ser5P), and that the histone acetyltransferase (HAT) subunit of SAGA (Gcn5) promotes increased histone acetylation, histone eviction, Pol II processivity, and histone H3-Lys4 methylation within coding sequences. We further showed that the histone H4 HAT complex, NuA4, is also recruited co-transcriptionally to coding regions via Ser5 phosphorylation of the Pol II CTD by the cyclin-dependent kinase (CDK) Cdk7/Kin28, and that NuA4 association with nucleosomes further depends on H3 methylation, presumably due to chromodomains and a PHD finger in NuA4 subunits. We obtained evidence that the HAT activities of NuA4 and SAGA cooperate to enhance co-transcriptional recruitment of the nucleosome remodeler RSC, promote histone eviction from transcribed sequences, and stimulate Pol II elongation. We subsequently extended the two-stage recruitment mechanism elucidated for NuA4, via Ser5P and methylated histones, to include the histone deacetylase complexes (HDACs) Rpd3C(S) and Set3C. Our results showed that, whereas interaction with methylated H3 is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated Pol II CTD (pCTD). We further demonstrated that the HDAs Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating nucleosomes and in limiting co-transcriptional nucleosome eviction, and provided strong evidence that histone acetylation is a key determinant of co-transcriptional nucleosome eviction. Our group also previously demonstrated that recruitment of the conserved transcription elongation factor Paf1C by Pol II is stimulated by elongation factor DSIF (comprised of Spt4 and Spt5) and the CDKs Cdk7/Kin28 and Cdk9/Bur1. We further showed that Ser5P CTD phosphorylation by Kin28 enhances recruitment of Bur1 near promoter regions via the pCTD-interaction domain (pCID) in the C-terminus of Bur1, and that Bur1 contributes to Serine-2 phosphorylation of the CTD (Ser2P) at the 5 ends of genes in addition to phosphorylating C-terminal repeats (CTRs) of Spt5. Subsequently, we established that Kin28 enhances Paf1C recruitment by promoting Bur1 recruitment via the Pol II pCTD with attendant phosphorylation of Spt5 CTRs, and by collaborating with Bur1 to generate Ser2-,Ser5-diphosphorylated CTD repeats that recruit Paf1C via pCIDs in Paf1C subunits. As pCIDs in Paf1C subunits also interact with phosphorylated Spt5 CTRs, we concluded that Pol II CTD repeats and Spt5 CTRs comprise distinct phosphorylated scaffolds that cooperate in Paf1C recruitment by elongating Pol II. Accumulation of a threonine biosynthetic intermediate attenuates general amino acid control by accelerating degradation of Gcn4 via the Cdks Pho85 and Cdk8/Srb10. Gcn4 abundance is tightly regulated by the interplay between the intricate translational control mechanism that induces Gcn4 synthesis in starved cells and a pathway of phosphorylation and ubiquitylation that mediates its rapid degradation by the proteasome, involving Cdks Pho85 and Srb10/Cdk8. By screening the yeast gene deletion library for mutations that impair transcriptional induction of amino acid biosynthetic genes by Gcn4, we discovered that transcriptional activation by Gcn4 is attenuated in mutants lacking the threonine biosynthetic enzyme encoded by HOM6, and determined that accumulation of the Hom6 substrate, beta-aspartate semialdehyde (ASA), is responsible for the reduced activation of Gcn4 target genes in hom6 mutant cells. Remarkably, ASA accumulation was found to accelerate the already rapid degradation of Gcn4 in a manner requiring its phosphorylation by Srb10 and Pho85. Our analysis of Gcn4 promoter occupancy and activation function of Gcn4 molecules rescued from degradation in srb10 versus pho85 mutant cells unveiled an unexpected division of labor between these two Cdks. Whereas Srb10 primarily targets inactive Gcn4 molecules that are presumably damaged under conditions of ASA excess, Pho85 clears a greater proportion of functional Gcn4 species from the cell. The ability of ASA to inhibit transcriptional induction of threonine pathway enzymes by Gcn4, dampening ASA accumulation and its toxic effects on other aspects cell physiology, is expected to be adaptive in the wild when yeast encounters natural antibiotics that target Hom6 enzymatic activity. Analysis of factors mediating nucleosome disassembly at Gcn4 target gene promoters in vivo. A key unsolved aspect of transcriptional activation by Gcn4 is the mechanism whereby Gcn4 binding to the UAS leads to eviction of nucleosomes that occlude promoter DNA sequences and block access by GTFs and Pol II. Certain nucleosome chaperones (Asf1, FACT subunits), heat-shock proteins with general chaperone activity, chromatin remodeling enzymes (SWI/SNF, ISWI, and Ino80 complexes) and histone deacetylases (Gcn5, Rtt109, Esa1) have been variously implicated in nucleosome disassembly at different yeast genes. It is unclear however whether the same ensemble of factors operates at all genes or whether each promoter is dependent on an idiosyncratic subset of factors, which might be dictated by the ability of the relevant transcriptional activator to recruit different factors to the promoter. We have explored the requirements for nucleosome chaperones, heat-shock proteins, chromatin remodeling complexes and HAT complexes for nucleosome disassembly in the promoters of 4 canonical Gcn4 target genes encoding amino acid biosynthetic enzymes. The results indicate that certain Gcn4 target promoters display a marked dependence on a particular factor for efficient nucleosome eviction, whereas other promoters are unaffected by the absence of that factor. However, many of these factors can be shown to promote nucleosome eviction at all 4 target genes by eliminating them from strains already lacking another factor. Thus, the factors under consideration contribute to nucleosome eviction at all four target genes but their functions overlap extensively with other factors at particular promoters. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-SEQ) to analyze histone and Pol II occupancies genome-wide, we recently determined that only a subset of Gcn4 target genes exhibit appreciable nucleosome eviction on transcriptional induction, which largley correspond to the 60 target genes that display the greatest induced occupancies of elongating Pol II. We are now examining the effects of eliminating different combinations of chaperones, chromatin remodeling complexes and HAT complexes on nucleosome disassembly at this large subset of genes to define the mechanism of nucleosome eviction for the Gcn4 transcriptome.
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