Transcriptional Regulation of Gene Expression

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We study the regulation of amino acid biosynthetic genes in budding yeast as a means of dissecting mechanisms of transcriptional control of gene expression, whose transcription 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 multiple cofactor complexes including SAGA, which contains the histone acetyltransferase (HAT) Gcn5, the chromatin remodeling (CR) complexes SWI/SNF and RSC, and the RNA Polymerase II (Pol II) Mediator, which in turn stimulate recruitment of general transcription factors (GTFs) and Pol II to the promoter to stimulate preinitiation complex (PIC) assembly. Seeking to identify all cofactors that mediate eviction of the -1 and +1 nucleosomes that occlude promoter DNA and impede binding of TBP and PIC assembly, we uncovered extensive cooperation among the chromatin remodeling complexes SWI/SNF, RSC, and Ino80C in evicting and repositioning promoter nucleosomes at genes activated by Gcn4 and other highly expressed genes in starved cells. We also showed that Ino80C acts in nucleosome eviction independently of its proposed function in replacing the specialized histone H2A.Z with conventional H2A in promoter nucleosomes. We further discovered that most of the occupied Gcn4 binding sites in the genome reside inside coding sequences (CDSs) rather than upstream of promoters, and that such non-canonical binding events frequently induce bidirectional transcription from within the coding sequences in addition to the conventional full-length mRNA transcripts of the gene. Distinct functions of three chromatin remodelers in activator binding and preinitiation complex assembly. As noted above, the CRs SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome depleted regions (NDRs) at the promoters of many yeast genes induced by Gcn4 during amino acid starvation. We analyzed mutants lacking the CR catalytic subunits for defects in binding of transcriptional activator Gcn4 and recruitment of TATA-binding protein (TBP) during preinitiation complex (PIC) assembly. RSC and Ino80 enhance Gcn4 binding to UAS elements in NDRs upstream of many promoters, and to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. Interestingly, SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at UAS elements, indicating a dual mode of action for this CR. All three CRs also stimulate TBP recruitment, at least partly by reducing nucleosome occupancies at TBP bindings sites, with SWI/SNF acting preferentially at the most highly expressed Gcn4 target genes. RSC and Ino80 function more broadly than SWI/SNF to stimulate TBP recruitment at most constitutively expressed genes, including ribosomal protein genes, whereas SWI/SNF acts preferentially at a distinct subset of highly expressed genes. These findings indicate a complex interplay among these three CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly (Rawal et al. 2022). Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes Previously, we showed that elimination of the HAT subunit in SAGA, Gcn5, did not fully impair nucleosome eviction at many starvation-induced genes, suggesting that Gcn5 might cooperate with other HATs in this process, similar to the functional cooperation we had identified for different CRs. The role of HAT complex NuA4, responsible for most H4 and H2A acetylation in yeast, was of particular interest as it was shown to be recruited to the Gcn4 target genes ARG1 and ARG4. We examined the effects of disrupting the NuA4 complex, by eliminating its nonessential scaffold subunit Eaf1, on promoter nucleosome eviction and transcriptional activation at both starvation-induced and constitutively expressed genes. We also examined whether depleting Eaf1 from the nucleus (by anchor-away technology) confers defects in nucleosome eviction or transcription in cells lacking Gcn5 to evaluate whether NuA4 and Gcn5 make independent, additive contributions to these processes at particular genes in vivo. Our results revealed that NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes, and in stimulating transcription, at starvation-induced genes. NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, recruitment of the TATA-binding protein (TBP), and transcription at most other genes expressed constitutively in yeast. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the highly expressed subset encoding ribosomal proteins (RPs), where Gcn5 contributes strongly to PIC assembly and transcription. We found that both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that appears to be controlled by their HAT activities, and thus most likely act directly to promote transcription of these genes. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes. Self-limiting activation through internal promoter activation by Gcn4 in amino acid-starved yeast. As noted above, functional Gcn4 binding sites often occur within gene CDSs rather than 5' of promoters, and that these internal Gcn4 binding sites (iGBSs) activate internal antisense transcripts (AS) in addition to the full-length (FL) sense transcripts of Gcn4 target genes. We hypothesize that Gcn4-induced AS transcription serves to limit Gcn4 induction of the FL transcripts via co-transcriptional histone methylation of the 5-promoters. Supporting this idea, an spt6-1004 mutation, known to reduce nucleosome densities in CDSs, was found to increase AS transcripts while repressing FL transcripts at many genes with iGBSs to a degree exceeding that found for genes lacking detectable AS transcripts. Eliminating Set1 and Set2, which mediate co-transcriptional histone methylation, also increases AS transcription at numerous genes with iGBSs, but without conferring a coupled reduction in FL transcripts. Importantly, deleting SET2 in the spt6-1004 mutant diminished repression of the FL transcripts without reducing AS transcription induced by spt6-1004. Chip-Seq analysis revealed increased H3K36 trimethylation of FL promoters in spt6-1004 cells to a greater extent at genes with iGBSs versus genes lacking detectable AS transcripts. These results support the model that AS transcription induced by internal Gcn4 binding increases histone methylation that limits activation of the upstream FL promoters by Gcn4. We are testing the possibility that this mechanism operates ubiquitously, including genes that fail to produce AS transcripts detectable in steady-state mRNA, by determining whether set2 suppresses the reduction in FL transcripts conferred genome-wide by spt6-1004.