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 recruitment of coactivator complexes Mediator, SAGA, SWI/SNF, and RSC, which collectively mediate nucleosome remodeling and recruitment of general transcription factors and RNA Polymerase II (Pol II) to promoters 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. 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 an evolutionarily conserved transcription elongation factor for Pol II required for regulating co-transcriptional histone methylation and optimal transcription rates in vivo. In addition to stimulating elongation, and enhancing recruitment of HDAs, Paf1C also promotes correct transcription termination in budding yeast. Moreover, the human Paf1C subunit parafibromin (hCdc73) is a known tumor suppressor. However, the mechanism of Paf1C recruitment by Pol II is not well understood. Previous work in our lab and others revealed that Paf1C recruitment is stimulated by the elongation factor DSIF (comprised of Spt4 and Spt5) and the CDKs Cdk7/Kin28 and Bur1, which both phosphorylate the Pol II CTD. Furthermore, it was known that Bur1 promotes Paf1C recruitment by phosphorylating the C-terminal hexad repeats (CTRs) in Spt5, but the underlying mechanism was unclear. The phosphorylated CTD (pCTD) is known to recruit mRNA processing enzymes and elongation/termination factors by elongating Pol II, and we showed previously that CTD phosphorylation on Serine-5 (S5P) by Kin28 enhances recruitment of Bur1 near promoter regions via the pCTD-interaction domain (pCID) in the C-terminus of Bur1. We further showed that Bur1 contributes to Serine-2 phosphorylation of the CTD (S2P) at the 5 ends of genes. Accordingly, Kin28 and Bur1 are predicted to collaborate in producing S2-,S5-diphosphorylated CTD repeats in promoter-proximal Pol II molecules. Our recent findings indicate that Kin28 enhances Paf1C recruitment in two ways. First, Kin28 promotes Bur1 recruitment via the pCTD with attendant phosphorylation of the Spt5 CTRs by Bur1. Second, S2-,S5-diphosphorylated CTD repeats produced in collaboration by Kin28 and Bur1 act directly in Paf1C recruitment via pCIDs in Paf1C subunits capable of recognizing these (and other) diphosphorylated CTD repeats. Interestingly, the pCIDs in certain Paf1C subunits also interact specifically with the phosphorylated forms of Spt5 CTRs. Hence, we propose that Pol II pCTD repeats and Spt5 pCTRs comprise distinct phosphorylated scaffolds in the Pol II elongation complex that cooperate to ensure high-level Paf1C recruitment by Pol II. This concerted mechanism might apply to other transcription cofactors known to rely on the pCTD for their recruitment by elongating 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 UAS-bound Gcn4 to stimulate PIC assembly and activate transcription. Recent work by our collaborator Vytas Bankaitis'group has 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 (eg. phytosphingosine (PHS)), in a manner dependent on sterol-binding protein Kes1 and the Mediator-associated CDK Srb10. Our contribution to this work was to demonstrate by chromatin IP analysis that PHS treatment reduces PIC assembly and Pol II occupancy at the Gcn4 target gene ARG1 to an extent that exceeds the decline in UAS occupancy by Gcn4 at this gene, thus suggesting a transcriptional defect of the type we uncovered previously in certain 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 increased cellular levels of SL as the result of aberrant expansion of the endosome. The resulting increase in SL levels provokes an Srb10-dependent impairment of Gcn4 activation function, blocking the GAAC response to amino acid starvation in sterol-deprived cells. Recently, we discovered unexpectedly that a number of Vps factors are associated with the coding sequences of Gcn4 and Gal4 target genes specifically under conditions of transcriptional activation and in a manner dependent on PIC assembly at the associated promoter region. Consistent with this, we uncovered marked defects in transcriptional elongation in a subset of vps mutants, most notably those lacking the PI(3)P kinase Vps34 and its associated protein kinase Vps15. Our collaborator Jiri Hasek has detected co-localization of fractions of Vps15 and Vps34 with the nuclear pore and transcription elongation factor Spt4. These findings suggest that certain Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed coding sequences in chromatin.

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3
Fiscal Year
2012
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$736,398
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Qiu, Hongfang; Chereji, Răzvan V; Hu, Cuihua et al. (2016) Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation. Genome Res 26:211-25
Ginsburg, Daniel S; Anlembom, Timi Elvuchio; Wang, Jianing et al. (2014) NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3. J Biol Chem 289:32656-70
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
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
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
Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G (2012) Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast. Methods Mol Biol 833:15-27
Qiu, Hongfang; Hu, Cuihua; Gaur, Naseem A et al. (2012) Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex. EMBO J 31:3494-505
Mousley, Carl J; Yuan, Peihua; Gaur, Naseem A et al. (2012) A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing. Cell 148:702-15
Jedidi, Iness; Zhang, Fan; Qiu, Hongfang et al. (2010) Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo. J Biol Chem 285:2438-55
Govind, Chhabi K; Qiu, Hongfang; Ginsburg, Daniel S et al. (2010) Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes. Mol Cell 39:234-46

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