Spt6 Is Required for the Fidelity of Promoter Selection

Abstract

SUMMARY Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters, and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation-site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Chromatin-sensitive cryptic promoters encode alternative protein isoforms in yeast

“…1 and 2). This is in line with the characterization of cryptic promoters in the chaperone mutant spt6-1004 that was published during the review of this manuscript (Doris et al 2018). Our MNase footprint analysis showed that those promoters present a canonical nucleosome array organization and suggested that canonical TFs bind upstream of the iTSSs in the body of genes and are associated to the appearance of intergenic NFR (Fig.…”

“…In fact, chromatin-sensitive iTSSs can also be detected, albeit at a much lower level, in wild-type conditions (see below). The Winston lab has recently investigated the appearance of intragenic promoters upon Spt6p depletion (spt6-1004) (Doris et al 2018). We compared up to what degree spt6-1004 up regulated intragenic promoters overlap with the chromatin-sensitive cryptic iTSS degined in this study ( Fig.…”

“…Specigically, interfering with the activity of the Rpd3S deacetylase complex, which recognizes Histone 3 Lys36 trimethylation 1 (H3K 36me3) deposited by t he histone methyltransferase Set2 during RNA polymerase II elongation, leads to intragenic cryptic transcription (Carrozza et al 2005;Lickwar et al 2009;Churchman and Weissman 2012;Chabbert et al 2015;Malabat et al 2015;Kim et al 2016). Likewise, modulating nucleosome positioning by impairing the function of histone chaperons such as Spt6p also leads to the appearance of intragenic cryptic transcripts (Kaplan et al 2003;Doris et al 2018). Spt6p depletion causes decreased expression of most genic promoters while increasing the expression of intragenic ones, thus suggesting a potential competition for initiation factors (Doris et al 2018).…”

“…Likewise, modulating nucleosome positioning by impairing the function of histone chaperons such as Spt6p also leads to the appearance of intragenic cryptic transcripts (Kaplan et al 2003;Doris et al 2018). Spt6p depletion causes decreased expression of most genic promoters while increasing the expression of intragenic ones, thus suggesting a potential competition for initiation factors (Doris et al 2018). In contrast, the second class of cryptic transcripts (RNA degradation-sensitive) are constitutively produced and degraded by the cell, and thus become detectable only when RNA degradation is impaired (Jensen et al 2013).…”

Auxin‐Inducible Degron System for Depletion of Proteins in Saccharomyces cerevisiae

“…Fourth, the degron tag described here uses a codon-optimized version of the AID tag and an OsTIR1 gene expressed under the control of the promoter of the yeast GPD1 gene, which ensures reliable and constitutive expression of TIR1 protein (Chan et al, 2018;Eng et al, 2014). Finally, the method is relatively simple and can be adapted to a variety of genomic and proteomic approaches for studying protein function (e.g., Doris et al, 2018;Gopalakrishnan, Marr, Kingston, & Winston, 2019;Shetty et al, 2017).…”