Combinatorial Genetic Control of Rpd3S through histone H3K4 and H3K36 Methylation in Budding Yeast

Abstract

1Much of euchromatin regulation occurs through reversible methylation of histone H3 2 lysine-4 and lysine-36 (H3K4me and H3K36me). Using the budding yeast Saccharomyces 3 cerevisiae, we previously found that levels of H3K4me modulated temperature sensitive alleles 4 of the transcriptional elongation complex Spt6-Spn1 through an unknown H3K4me effector 5 pathway. Here we identify the Rpd3S histone deacetylase complex as the H3K4me effector 6 underlying these Spt6-Spn1 genetic interactions. Exploiting these Spt6-Spn1 genetic interactions, 7we show that H3K4me and H3K36me collaboratively impact Rpd3S function in an opposing 8 manner. H3K36me is deposited by the histone methyltransferase Set2 and is known to promote 9Rpd3S function at RNA PolII transcribed open reading frames. Using genetic epistasis 10 experiments, we find that mutations perturbing the Set2-H3K36me-Rpd3S pathway suppress the 11 growth defects caused by temperature sensitive alleles of SPT6 and SPN1, illuminating that this 12 pathway antagonizes Spt6-Spn1. Using these sensitive genetic assays, we also identify a role for 13H3K4me in antagonizing Rpd3S that functions through the Rpd3S subunit Rco1, which is known 14 to bind H3 N-terminal tails in a manner that is prevented by H3K4me. Further genetic 15 experiments reveal that the H3K4 and H3K36 demethylases JHD2 and RPH1 mediate this 16 combinatorial control of Rpd3S. Finally, our studies also show that the Rpd3L complex, which 17 acts at promoter-proximal regions of PolII transcribed genes, counters Rpd3S for genetic 18 modulation of Spt6-Spn1, and that these two Rpd3 complexes balance the activities of each 19 other. Our findings present the first evidence that H3K4me and H3K36me act combinatorially to 20 control Rpd3S. 21 22

Essential histone chaperones collaborate to regulate transcription and chromatin integrity

“…Analysis of the 17 extragenic suppressors identified mutations in seven genes, all of which encode factors that regulate transcription elongation and chromatin ( Figure 2A). Four of these genes -SET2, RCO1, CHD1, and HTA1 -were previously identified as suppressors of either spt6 mutations or mutations that impair related elongation factors (Chu et al, 2006;Keogh et al, 2005;Lee et al, 2018;McCullough et al, 2015;Quan and Hartzog, 2010). As three of the genes, SET2, RCO1, and CHD1, are nonessential for viability, we tested complete deletions of each and found that they also suppressed spt6-YW, showing that suppression is conferred by loss of function (Table S2).…”

“…Spt16 and Pob3 are the two subunits of the histone chaperone FACT (Gurova et al, 2018), and Spt5 is a conserved elongation factor that interacts directly with RNAPII and has recently been implicated in controlling chromatin structure (Crickard et al, 2017;Ehara et al, 2019;Hartzog and Fu, 2013). The identification of spt6-YW suppressors in SPT16, POB3, and SPT5 was unexpected, as previously isolated mutations in these genes conferred similar, rather than opposite phenotypes as spt6 mutations (Cheung et al, 2008;Hainer et al, 2011;Hartzog et al, 1998;Jeronimo et al, 2019;Jeronimo et al, 2015;Kaplan et al, 2003;Lee et al, 2018;Malone et al, 1991;McCullough et al, 2015;Morillo-Huesca et al, 2010;Pathak et al, 2018;Swanson and Winston, 1992). The analysis of the FACT and Spt5 suppressors is the focus of the rest of these studies.…”

Suppressor mutations that make the essential transcription factor Spn1/Iws1 dispensable in Saccharomyces cerevisiae

“…2015) or that impair other chromatin-related factors, including FACT, Swi/Snf, Set2, and Rpd3S (Z hang et al . 2008; L ee et al . 2018b; V iktorovskaya et al .…”

Suppressor mutations that make the essential transcription factor Spn1/Iws1 dispensable inSaccharomyces cerevisiae

“…Second, in vivo, a temperature-sensitive spn1 mutation, spn1-K192N (FISCHBECK et al 2002), causes changes in chromatin structure, resulting in altered spacing between nucleosomes and increased nucleosome fuzziness (VIKTOROVSKAYA et al 2021). Third, spn1-K192N is suppressed by mutations that alter histone H2A (MCCULLOUGH et al 2015) or that impair other chromatinrelated factors, including FACT, Swi/Snf, Set2, and Rpd3S (ZHANG et al 2008;LEE et al 2018b;VIKTOROVSKAYA et al 2021). Finally, spn1 mutations cause double mutant sickness or lethality when combined with mutations that impair other histone chaperones (LI et al 2018) or histone H3K4 methylation (LEE et al 2018a).…”