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 activator Gcn4 during amino acid limitation. We and others showed previously that transcriptional activation by Gcn4 is enhanced by its recruitment of cofactor 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. A key unsolved aspect of transcriptional activation by Gcn4 is how it mediates the eviction of the -1 and +1 nucleosomes that occlude promoter DNA and block access by GTFs and Pol II. Indeed, the mechanism of this key step of gene activation is not fully understood for any yeast genes. Previous studies implicated histone chaperones, chromatin remodelers or histone acetyltransferases in the process at particular genes, but it was unclear whether these co-factors function universally in promoter nucleosome eviction, as co-factor requirements at most yeast promoters are unknown. We are addressing the mechanism of nucleosome eviction and the consequences of defects in this process on transcription for the hundreds of co-regulated genes in the Gcn4 transcriptome as well as for all other constitutively expressed genes. Using H3 chromatin immunoprecipitation (ChIP) coupled to deep-sequencing (H3 ChIP-Seq) of wild-type and mutant yeast strains lacking Snf2, Gcn5 and Ydj1, we established that these cofactors collaborate in evicting H3 from the -1 and +1 nucleosomes, and intervening nucleosome-depleted region (NDR) at a large fraction of Gcn4 target genes. Moreover, these three cofactors were found to cooperate similarly at the majority of all other 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 comprised of the induced Gcn4 transcriptome and most highly expressed constitutively expressed yeast genes, whereas the most weakly expressed genes displayed an increase in relative transcription. Thus, we established that eviction of promoter nucleosomes is required for maximal transcription of highly expressed genes, and Gcn5, Snf2, and Ydj1 function broadly in this step of gene activation, while some other aspect of transcriptional activation is more generally rate-limiting for transcription of most genes in amino acid-deprived yeast. SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes 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 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. Gcn4 binding within coding regions can activate both internal and canonical 5 promoters in yeast We are also interested in determining the role of promoter nucleosome eviction in controlling binding of Gcn4 itself upstream from the promoters of its target genes, and set out to define all of the binding sites for Gcn4 throughout the genome in WT cells. ChIP-seq analysis of Gcn4 binding revealed 546 genomic sites occupied by Gcn4 in starved cells, representing only 30% of all genomic sequences with significant matches to the consensus Gcn4 binding motif. Analysis of nucleosome occupancy data from MNase-seq analysis revealed that distance of a motif from the nearest nucleosome dyad and its match to the consensus sequence are the major determinants of Gcn4 binding in vivo. Surprisingly, only 40% of the bound sites are in promoters, and analysis of genome-wide mRNA expression data and ChIP-seq analysis of RNA Pol II in starvation conditions indicates that only 60% of such promoter-located Gcn4 occupancy peaks activate transcriptionindicating extensive negative control over Gcn4 function. Remarkably, most of the remaining 300 Gcn4-bound motifs reside within coding sequences (CDS), with 75 representing the only bound motif in the vicinity of a known Gcn4-induced gene. RNA-seq analysis revealed that many such unconventional Gcn4 occupancy peaks map between divergent antisense and sub-genic sense transcripts induced from within CDS under starvation conditions, and are also located adjacent to starvation-induced TBP occupancy peaks detected by ChIP-seq analysis. These findings are consistent with Gcn4 activation of cryptic, bidirectional internal promoters at these genes. Mutational analysis confirmed that Gcn4-bound motifs within CDS can activate both sub-genic and full-length transcripts from the same or adjacent genes, demonstrating that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by a well-defined activator that also functions at conventional 5-positioned promoters.
Showing the most recent 10 out of 17 publications
