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. 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 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. An unsolved question is how Gcn4 mediates eviction of the -1 and +1 nucleosomes that occlude promoter DNA. By histone H3 chromatin immunoprecipitation-sequencing (H3 ChIP-Seq) of wild-type and mutants lacking the catalytic subunit of SWI/SNF (Snf2), Gcn5 and the Hsp70 co-chaperone Ydj1, we previously established that these cofactors participate in evicting promoter nucleosomes, and in repositioning non-evicted -1 and +1 nucleosomes to increase NDR widths, to expose promoter DNA and enhance transcription of many Gcn4-induced genes and other genes expressed constitutively at high levels. Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes. The CR Ino80C has been implicated in replacing the histone variant H2A.Z (encoded by HTZ1 and enriched at the +1 nucleosome) with canonical H2A. Removal of an H2A.Z:H2B dimer by Ino80C could render the partially disassembled nucleosome more susceptible to eviction. By ChIP-seq analysis of H3 and Pol II in an ino80 mutant lacking the Ino80C catalytic subunit, we found that Ino80C functions on par with SWI/SNF in eviction of promoter nucleosomes and transcriptional activation of Gcn4 target genes, and plays a much greater role than SWI/SNF at several hundred Ino80C-hyperdependent genes. At Gcn4 target genes, the degree of nucleosome eviction defect is correlated with a reduction in transcription and in promoter occupancies of the key GTF TATA-binding protein (TBP). ChIP-seq analysis of Ino80 shows that Ino80C is enriched at both Gcn4 target genes and Ino80C-hyperdependent genes, indicating a direct role for this CR in evicting promoter nucleosomes to enhance PIC assembly and transcription at highly expressed genes (Qiu et al., 2020). If Ino80C enhances nucleosome eviction strictly in the course of editing H2A.Z-H2B dimers, then deleting HTZ1 should mimic the effect of deleting INO80 on promoter nucleosome eviction. Moreover, depleting Ino80 should have no effect on nucleosome occupancies in cells lacking HTZ1. At odds with these predictions, deleting HTZ1 has a far smaller effect than eliminating INO80 on eviction of promoter nucleosomes. Moreover, nuclear depletion of Ino80 from the nucleus (by anchor-away) impaired histone eviction even in cells lacking HTZ1. Thus, Ino80C can function like the SWI/SNF family members, SWI/SNF and RSC, in promoting chromatin access independently of nucleosome editing (Qiu et al., 2020). Gcn4 binding within coding regions activates both internal and canonical 5 promoters in yeast We are interested in determining the role of promoter nucleosome eviction in modulating binding of Gcn4 itself at its target genes, and used Gcn4 ChIP-seq to identify all of the Gcn4 binding sites in the genome. Surprisingly, only 40% of the 540 Gcn4-bound sites are in promoters, and only 60% of such sites appear to activate transcription, indicating extensive negative control over Gcn4 function. 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 (AS) 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. Thus, internal promoters can be regulated by a well-defined activator like Gcn4 that also functions at conventional 5-promoters. Current experiments are aimed at determining whether induction of internal AS promoters by Gcn4 serves to dampen its activation of the upstream promoters for the full-length sense transcripts at these genes, via collisions between Pol II molecules transcribing from opposite DNA strands, or by co-transcriptional methylation by Set1 or Set2 with attendant deacetylation of the upstream promoter nucleosomes. We are also examining the roles of Set1 and Set2, and the histone chaperone Spt6 in suppressing cryptic internal promoters activated by Gcn4, and of the nuclear exosome and the nonsense-mediated-decay (NMD) pathway in degradation of the AS transcripts. 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.