Chhabi K. Govind

Phosphorylated Pol II CTD Recruits Multiple HDACs, Including Rpd3C(S), for Methylation-Dependent Deacetylation of ORF Nucleosomes

Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction.

Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo.

ABSTRACT Transcriptional activation by Gcn4p is dependent on the coactivators SWI/SNF, SAGA, and Srb Mediator, which are recruited by Gcn4p and stimulate assembly of the preinitiation complex (PIC) at the ARG1 promoter in vivo. We show that recruitment of all three coactivators is nearly simultaneous with binding of Gcn4p at ARG1 and is followed quickly by PIC formation and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (Gcn5p), a non-HAT function of SAGA, and on Mediator. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1. We confirm the roles of Mediator and SAGA in TATA binding protein (TBP) recruitment and demonstrate that all four coactivators under study enhance Pol II recruitment or promoter clearance following TBP binding. We also present evidence that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. These functions can be limited to discrete time intervals, providing evidence for multiple stages in the induction process. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes Gcn4p from other yeast activators studied thus far.

Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2.

Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

ABSTRACT NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4s interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Activator Gcn4p and Cyc8p/Tup1p Are Interdependent for Promoter Occupancy at ARG1 In Vivo

ABSTRACT The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

A role for phosphorylated Pol II CTD in modulating transcription coupled histone dynamics.

Histone acetylation modulates histone occupancy both at promoters and in coding sequences. Based on our recent observation that HDACs in the budding yeast, Saccharomyces cerevisiae, are co-transcriptionally recruited to coding regions by elongating polymerases, we propose a model in which Pol II facilitates recruitment of chromatin remodeling complexes as well as other factors required for productive elongation.

Measuring Dynamic Changes in Histone Modifications and Nucleosome Density during Activated Transcription in Budding Yeast

Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

Recruitment of Saccharomyces cerevisiae Cmr1/Ydl156w to Coding Regions Promotes Transcription Genome Wide

Cmr1 (changed mutation rate 1) is a largely uncharacterized nuclear protein that has recently emerged in several global genetic interaction and protein localization studies. It clusters with proteins involved in DNA damage and replication stress response, suggesting a role in maintaining genome integrity. Under conditions of proteasome inhibition or replication stress, this protein localizes to distinct sub-nuclear foci termed as intranuclear quality control (INQ) compartments, which sequester proteins for their subsequent degradation. Interestingly, it also interacts with histones, chromatin remodelers and modifiers, as well as with proteins involved in transcription including subunits of RNA Pol I and Pol III, but not with those of Pol II. It is not known whether Cmr1 plays a role in regulating transcription of Pol II target genes. Here, we show that Cmr1 is recruited to the coding regions of transcribed genes of S. cerevisiae. Cmr1 occupancy correlates with the Pol II occupancy genome-wide, indicating that it is recruited to coding sequences in a transcription-dependent manner. Cmr1-enriched genes include Gcn4 targets and ribosomal protein genes. Furthermore, our results show that Cmr1 recruitment to coding sequences is stimulated by Pol II CTD kinase, Kin28, and the histone deacetylases, Rpd3 and Hos2. Finally, our genome-wide analyses implicate Cmr1 in regulating Pol II occupancy at transcribed coding sequences. However, it is dispensable for maintaining co-transcriptional histone occupancy and histone modification (acetylation and methylation). Collectively, our results show that Cmr1 facilitates transcription by directly engaging with transcribed coding regions.

Saccharomyces cerevisiae histone Chaperones FACT and Spt6 modulate Pol II and histone occupancy genome-wide

Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, FACT and Spt6, in regulating transcription and histone occupancy. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 HAT Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT-depletion greatly reduces Pol II occupancy in the 5’ ends genome-wide. By contrast, Spt6-depletion led to reduction in Pol II occupancy towards the 3’ end, in a manner dependent on the gene-length. Severe transcription and histone eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6Δ202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with WT Pol II occupancies at these genes, indicating critical roles of Spt6 and Spt16 in promoting high-level transcription. Collectively, our study shows cooperation, as well as redundancy between chaperones, FACT and Spt6, in regulating transcription and chromatin in coding regions of transcribed genes.