We study the regulation of amino acid and vitamin 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 in response to 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 showed that the histone H4 HAT complex, NuA4, is also recruited co-transcriptionally to coding regions via Ser5 phosphorylation by CTD kinase 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. Thus, inactivating Gcn5 and the HAT subunit in NuA4 (Esa1) confers additive reductions in transcript production from long versus short coding sequences, and reduces the rate of Pol run-off from an extended coding sequence following promoter shut-off. These findings demonstrated direct, additive roles for the HAT activities of NuA4 and SAGA in promoting the elongation phase of Pol II transcription. We recently extended the two-stage recruitment mechanism, via Ser5P and methylated histones, described above for NuA4 to include the histone deacetylase complex (HDAC) Rpd3C(S). It was known that methylation of H3 by Set1 and Set2 is required for deacetylation of coding region nucleosomes by Set3C and Rpd3C(S), respectively. We discovered that Set3C and Rpd3C(S) are co-transcriptionally recruited to coding sequences in the absence of both Set1 and Set2, but in a manner stimulated by Cdk7/Kin28. Moreover, Rpd3C(S) and Set3C were shown to interact with both Pol II-Ser5P and histones in extracts, but only the histone interactions require H3 methylation. Moreover, a reconstituted Rpd3C(S) complex bound specifically to Ser5P synthetic peptides. Thus, 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 limiting co-transcriptional nucleosome eviction. Moreover, a strong correlation between increased acetylation and lower histone occupancy observed in single, double, and triple HDA mutants supports our contention, based on analysis of HAT mutants, that histone acetylation is a key determinant of co-transcriptional nucleosome eviction. Previously, we showed that the cyclin-dependent kinase (Cdk) Bur1/Bur2 is recruited to promoters through direct interaction with Ser5-phosphorylated Pol II CTD (generated by Kin28), phosphorylates Ser2 in CTD repeats near the promoter, and also stimulates Ser2-CTD phosphorylation at promoter-distal sites by the Cdk Ctk1. Recently, it was demonstrated that Bur1 also phosphorylates C-terminal repeats (CTRs) of elongation factor Spt5 in a manner that facilitates co-transcriptional recruitment of the elongation factor Paf1C. We are currently pursuing recent findings indicating that Paf1C recruitment occurs by a dual mechanism involving direct binding by three Paf1C subunits to Pol II CTD repeats diphosphorylated on Ser5 and Ser2 (by Kin28 and Bur1) in addition to Spt5 CTRs phosphorylated by Bur1. The Mediator is a multisubunit coactivator required for transcription initiation by Pol II, which is recruited by Gcn4 to its target promoters in vivo. Previously, we determined that the tail subdomain of Mediator, containing the Gal11/Med15 subunit, is a direct target of Gcn4 in vivo, critical for recruitment by Gcn4 of both intact Mediator and the stable Mediator tail subdomain existing in sin4-del mutant cells. Although several Gal11 segments were shown previously to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We demonstrated that interaction of Gcn4 with the Mediator tail in vitro, and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo, involve additive contributions from three different segments in the N-terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we established that the B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrated that Gcn4 binds to the Gal11 KIX domain directly and, in collaboration with Christopher Jaroniecs group at Ohio State University, utilized NMR chemical shift analysis, combined with mutational studies, to identify the likely Gcn4 binding site on the surface of the KIX domain. Together, these results define the physiological mechanism of Mediator recruitment by Gcn4. It appears that Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator to target promoters in vivo. 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 Vytas Bankaitis group at UNC-Chapel Hill 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 ARG1 to an extent that exceeds the decline in UAS occupancy by Gcn4 at this gene, suggesting a transcriptional defect of the type we described 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.

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Rawal, Yashpal; Chereji, R?zvan V; Valabhoju, Vishalini et al. (2018) Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast. Mol Cell 70:297-311.e4
Rawal, Yashpal; Chereji, R?zvan V; Qiu, Hongfang et al. (2018) SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast. Genes Dev 32:695-710
Qiu, Hongfang; Chereji, R?zvan 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
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
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

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