We study the regulation of amino acid biosynthetic genes in budding yeast as a means of dissecting mechanisms of transcriptional control of gene expression. Transcription of these genes is coordinately induced by the activator Gcn4 during amino acid limitation--the general amino acid control (GAAC)owing to accumulation of Gcn4 in starved cells. We showed previously that transcriptional activation by Gcn4 is enhanced by its recruitment of coactivator complexes Mediator, SAGA, SWI/SNF, and RSC, which in turn stimulate 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 (CDS) by association with the Ser5-phosphorylated C-terminal domain (CTD) of Pol II, 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 CDS. We showed that histone H4 HAT complex, NuA4, is also recruited co-transcriptionally via Ser5 phosphorylation of the Pol II CTD by cyclin-dependent kinase (CDK) Cdk7/Kin28, and that NuA4 association with nucleosomes also depends on H3 methylation, presumably due to chromodomains and a PHD finger in NuA4 subunits. We obtained evidence that HAT activities of NuA4 and SAGA cooperate to enhance co-transcriptional recruitment of nucleosome remodeling complex RSC, promote histone eviction from transcribed CDS, and stimulate Pol II elongation. We subsequently extended the two-stage recruitment mechanism elucidated for NuA4, via Ser5P and methylated histones, to include histone deacetylase complexes (HDACs) Rpd3C(S) and Set3C. Our results revealed that, whereas interaction with methylated H3 is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phospho-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 evidence that histone acetylation is a key determinant of co-transcriptional nucleosome eviction. More recently, we provided evidence that NuA4 recruitment is stimulated by methylation of Lys4 and Lys36 in the H3 tail by Set1 and Set2, and that NuA4 acetylation of H4 tail lysines stimulates SAGA recruitment and attendant acetylation of H3 in vivo. Thus, H3 methylation exerts opposing effects of enhancing nucleosome acetylation by NuA4 and SAGA while stimulating nucleosome deacetylation by HDACs to maintain the proper level of histone acetylation in transcribed genes. Moreover, recruitment of one HAT complex (NuA4) was found to enhance that of another (SAGA) with complementary histone substrate specificity. Analysis of factors mediating nucleosome disassembly at Gcn4 target gene promoters in vivo. A key unsolved aspect of transcriptional activation by Gcn4 is how it mediates the eviction of nucleosomes that occlude promoter DNA sequences and block access by GTFs and Pol II. Indeed, the mechanism of this key step of gene activation, and the impact of defective nucleosome eviction on transcription, are not fully understood for any yeast genes. Previous studies implicated certain histone chaperones, chromatin remodelers or histone acetyltransferases in the remodeling or eviction of nucleosomes from the promoters of certain yeast genes, but it was unclear whether these co-factors function broadly in nucleosome eviction, as co-factor requirements at most yeast promoters are unknown. Eviction of promoter nucleosomes is considered to be rate-limiting for transcriptional activation, but the consequences of impaired nucleosome eviction on transcription have not been analyzed genome-wide. We addressed these questions by analyzing histone H3 eviction for the hundreds of genes in the Gcn4 transcriptome on induction of Gcn4 in a large panel of mutants lacking one or more co-factors implicated at particular yeast genes. By conventional chromatin immunoprecipitation analysis (ChIP) of four canonical Gcn4 target genes, ARG1, HIS4, ARG4, and CPA2, we excluded a requirement for several co-factors implicated previously at other genes (eg. Asf1, Nap1, RSC) and implicated the remodeler SWI/SNF (Snf2), HAT Gcn5, and Hsp70 co-chaperone Ydj1 in nucleosome eviction at these Gcn4 target genes. Expanding our analysis genome-wide by H3 ChIP-Seq, we found that Snf2, Gcn5 and Ydj1 collaborate in evicting H3 from the -1 and +1 promoter nucleosomes, and intervening nucleosome-depleted region (NDR) at a large fraction of the Gcn4 transcriptome. These 3 cofactors were found to function similarly at virtually all yeast promoters. Surprisingly, however, defective H3 eviction in co-factor mutants was coupled with reduced transcription (Pol II densities measured by Rpb3 ChIP-Seq) for only a subset of genes, which included the induced Gcn4 transcriptome and the most highly expressed subset of constitutively expressed yeast genes. In fact, the most weakly expressed genes displayed an increase in transcription relative to other genes in response to global attenuation of nucleosome eviction. Thus, we established that steady-state eviction of promoter nucleosomes is required for maximal transcription of highly expressed genes, and that Gcn5, Snf2, and Ydj1 function broadly in this step of gene activation, but discovered unexpectedly that some other aspect of transcriptional activation is more generally rate-limiting for transcription of most genes in amino acid-deprived yeast. SWI/SNF partners with RSC to reposition and evict promoter nucleosomes for transcriptional activation at highly expressed genes in yeast Having observed previously that nucleosome eviction in the induced Gcn4 transcriptome is only partially impaired in cells lacking Snf2, we surmised that SWI/SNF cooperates with one or more other remodeling factors in evicting promoter nucleosomes. Considering that RSC and SWI/SNF belong to the same family of remodeling complexes, we asked whether SWI/SNF and RSC cooperate in nucleosome eviction at genes induced by Gcn4, and also at genes expressed constitutively at high levels where previously we found that SWI/SNF cooperates with Gcn5 and Ydj1. We also explored whether SWI/SNF resembles RSC in determining the positions of -1 and +1 nucleosomes and hence, NDR width, at highly expressed genes. Our findings thus far reveal a previously undetected widening of NDRs, in addition to eviction of the -1 and +1 nucleosomes, on induction of Gcn4 target genes in WT cells, and demonstrate that SWI/SNF and RSC have distinct and equally critical roles in achieving wide, nucleosome-depleted NDRs for robust transcription at these induced genes. We also uncovered cooperation between SWI/SNF and RSC in nucleosome positioning and eviction at the most highly transcribed subset of constitutively expressed genes, suggesting their general cooperation in achieving high transcription rates. The occupancies of both remodelers were found to be greatest at highly expressed or induced genes, supporting direct functions for both remodelers at this group of genes. Our results reveal an extensive division of labor between SWI/SNF and RSC in promoter nucleosome eviction and displacement at the most highly transcribed genes in yeast.
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