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 the response known as general amino acid control (GAAC). Gcn4 expression is coupled to amino acid levels through a translational control mechanism that increases the 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 the recruitment of general transcription factors and RNA Polymerase II (Pol II) to the promoter for stimulating 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. Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex. The Paf1 complex (Paf1C) is a conserved transcription elongation factor for Pol II. Work done in our lab and others showed that Paf1C recruitment is stimulated by elongation factor DSIF (comprised of Spt4 and Spt5) and the CDKs Cdk7/Kin28 and Bur1, which both phosphorylate the Pol II CTD. We also showed that Ser5P CTD phosphorylation by Kin28 enhances recruitment of Bur1 near promoter regions via the pCTD-interaction domain (pCID) in the Bur1 C-terminus, and that Bur1 contributes to Serine-2 phosphorylation of the CTD (Ser2P) at the 5'ends of genes. Thus, Kin28 and Bur1 collaborate in producing Ser2-,Ser5-diphosphorylated CTD repeats in promoter-proximal Pol II molecules. Our recent findings provide evidence that Kin28 enhances Paf1C recruitment in two ways, by promoting Bur1 recruitment via the pCTD with attendant phosphorylation of Spt5 CTRs by Bur1, and by collaborating with Bur1 to generate Ser2-,Ser5-diphosphorylated CTD repeats that recruit Paf1C via pCIDs in Paf1C subunits. Interestingly, the pCIDs in certain Paf1C subunits also interact with phosphorylated Spt5 CTRs. Hence, we propose that Pol II pCTD repeats and Spt5 pCTRs comprise distinct phosphorylated scaffolds in the elongation complex that cooperate to ensure high-level Paf1C recruitment by Pol II. Roles of vacuolar protein sorting (Vps) factors in regulating transcription. Previously, we reported that disruption of vesicular protein transport at the late endosome/MVB occurring in a subset of vps mutants impedes the ability of nucleus-localized and promoter-bound Gcn4 to stimulate PIC assembly. Recent work in the Bankaitis group led to the model that sterol limitation, or defects in vesicular protein trafficking, provoke a block in Gcn4 function that is triggered by accumulation of sphingolipids (SL)in a manner dependent on sterol-binding protein Kes1 and the Mediator-associated CDK Srb10. We demonstrated that SL treatment reduces PIC assembly and Pol II occupancy at the Gcn4 target gene ARG1 to an extent that exceeds the decline in Gcn4 promoter occupancy, suggesting a transcriptional defect similar to that seen in vps mutants. Bankaitis et al propose that Kes1 provides a sterol-regulated brake on vesicular protein transport and that increased Kes1 binding to endomembranes at low sterol levels evokes SL accumulation as the result of aberrant endosome expansion. The elevated SL provokes Srb10-dependent impairment of Gcn4 activation function, blocking the GAAC response to amino acid starvation in sterol-deprived cells. We discovered unexpectedly that various Vps factors are associated with the coding sequences of Gcn4 and Gal4 target genes specifically under conditions of transcriptional activation and dependent on PIC assembly at the associated promoters. Consistent with this, we uncovered defects in transcriptional elongation in a subset of vps mutants, most notably those lacking PI(3)P kinase Vps34 and its associated protein kinase Vps15. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with genetic interactions with mutations of the histone acetyltransferase complex NuA4, vps15 and vps34 mutations reduce NuA4 occupancy in certain transcribed CDS. vps15 and vps34 mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery, and we observed co-localization of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin. Analysis of factors mediating nucleosome disassembly at Gcn4 target gene promoters. An unsolved aspect of transcriptional activation by Gcn4 is its mechanism for eviction of nucleosomes that block access by GTFs and Pol II to the promoter. Nucleosome chaperones (Asf1, FACT), heat-shock proteins, chromatin remodeling enzymes (SWI/SNF, ISWI, Ino80) and histone deacetylases (Gcn5, Rtt109, Esa1) have been implicated variously in nucleosome disassembly at different genes, but it is unclear whether the same ensemble of factors operates at all genes or whether each promoter uses an idiosyncratic subset of factors dictated by the properties of the activator or promoter sequence/chromatin structure. We are exploring the factor requirements for nucleosome disassembly in the promoters of canonical Gcn4 target genes encoding amino acid biosynthetic enzymes. Our results thus far indicate that certain Gcn4 target promoters display a marked dependence on a given factor for 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 target genes tested by eliminating them from strains already lacking a second factor. These data suggest that the factors under consideration contribute to nucleosome eviction at every Gcn4 target gene but that their functions overlap extensively at certain promoters.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2013
Total Cost
$472,220
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Rawal, Yashpal; Qiu, Hongfang; Hinnebusch, Alan G (2014) Accumulation of a Threonine Biosynthetic Intermediate Attenuates General Amino Acid Control by Accelerating Degradation of Gcn4 via Pho85 and Cdk8. PLoS Genet 10:e1004534
Kong, Ka-Yiu Edwin; Tang, Hei-Man Vincent; Pan, Kewu et al. (2014) Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing. Nucleic Acids Res 42:643-60
Gaur, Naseem A; Hasek, Jiri; Brickner, Donna Garvey et al. (2013) Vps factors are required for efficient transcription elongation in budding yeast. Genetics 193:829-51