Combinatorial Genetic Control of Rpd3S Through Histone H3K4 and H3K36 Methylation in Budding Yeast

Abstract

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

The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae

“…By this model, H3K4me3 would inhibit deacetylation by Rpd3S in the region immediately downstream of promoters, where H3K4me3 is primarily found ( 37–41 ). In line with the idea that H3K36 methylation and H3K4 methylation have opposing functions, studies showed that loss of H3K4 methylation exacerbated the Ts − phenotype of spn1-K192N , while increased levels of H3K4 methylation suppressed spn1-K192 N ( 21 , 22 ). In summary, genetic analysis suggests that the Set2-Rpd3S pathway opposes the function of Spn1, while Set1-dependent H3K4 methylation promotes it.…”

“…In contrast to the activation of Rpd3S by H3K36 methylation, Set1-dependent H3K4 methylation has been proposed to oppose the function of Rpd3S, similar to the role proposed for Spn1 ( 22 , 36 ). By this model, H3K4me3 would inhibit deacetylation by Rpd3S in the region immediately downstream of promoters, where H3K4me3 is primarily found ( 37–41 ).…”

“…Spn1 directly interacts with Spt6 ( 17 , 18 ), another essential histone chaperone that associates with elongating RNAPII ( 10 , 19 , 20 ), and some evidence suggests that the two factors contribute to a common function. For example, some spn1 and spt6 mutations exhibit similar genetic interactions with mutations in genes encoding other factors involved in transcription and chromatin dynamics ( 21 , 22 ). In addition, combining spn1 and spt6 mutations or disrupting the Spn1-Spt6 interaction by mutation results in synthetic lethality, poor growth, or phenotypes associated with defective transcription and chromatin organization ((17,18,23) and our unpublished results).…”

“…A functional connection between Spn1 and H3K36 methylation in yeast came from recent genetic evidence that showed that null mutations in genes encoding Set2 or components of the Rpd3S histone deacetylase complex ( set2Δ , rpd3Δ , eaf3Δ , rco1Δ ) suppress the temperature sensitive phenotype of a spn1 mutation, spn1-K192N ( 22 ). Rpd3S is recruited and activated by H3K36me2/3 and the Ser5-phosphorylated RNAPII CTD ( 24 , 31–35 ).…”

“…Rpd3S is recruited and activated by H3K36me2/3 and the Ser5-phosphorylated RNAPII CTD ( 24 , 31–35 ). This suppression of spn1-K192N ( 22 ) suggested that one role of Spn1 is to oppose the functions of the Rpd3S histone deacetylase complex and, furthermore, that increased histone acetylation can compensate for impairment of Spn1 function.…”