Role of Mediator in regulating Pol II elongation and nucleosome displacement in Saccharomyces cerevisiae

Abstract

Mediator is a modular multisubunit complex that functions as a critical coregulator of RNA polymerase II (Pol II) transcription. While it is well accepted that Mediator plays important roles in the assembly and function of the preinitiation complex (PIC), less is known of its potential roles in regulating downstream steps of the transcription cycle. Here we use a combination of genetic and molecular approaches to investigate Mediator regulation of Pol II elongation in the model eukaryote, Saccharomyces cerevisiae. We find that ewe (expression without heat shock element) mutations in conserved Mediator subunits Med7, Med14, Med19, and Med21-all located within or adjacent to the middle module-severely diminish heat-shock-induced expression of the Hsf1-regulated HSP82 gene. Interestingly, these mutations do not impede Pol II recruitment to the gene's promoter but instead impair its transit through the coding region. This implies that a normal function of Mediator is to regulate a postinitiation step at HSP82. In addition, displacement of histones from promoter and coding regions, a hallmark of activated heat-shock genes, is significantly impaired in the med14 and med21 mutants. Suggestive of a more general role, ewe mutations confer hypersensitivity to the anti-elongation drug 6-azauracil (6-AU) and one of them-med21-impairs Pol II processivity on a GAL1-regulated reporter gene. Taken together, our results suggest that yeast Mediator, acting principally through its middle module, can regulate Pol II elongation at both heat-shock and non-heat-shock genes.

Figure 1

Growth phenotypes of the MED⁺ strain SK-HS1004 and congenic ewe mutants employed in this study. Cells were grown on rich medium at the indicated temperature for 2 days.

Figure 2

ewe mutations diminish both noninduced and induced HSP82 transcription. (A) RT–qPCR analysis of basal HSP82 mRNA expression levels in the MED⁺ strain and ewe mutant derivatives. Cells were cultivated at 30° and RNA was isolated. HSP82 mRNA/SCR1 RNA quotients are shown. (SCR1 is a Pol III transcript and served as an internal recovery control.) The MED⁺ quotient was set to 1.0, and all others were normalized to it. (B) RT–qPCR analysis as in A, except cells were heat-shocked for 2, 15, or 60 min and both HSP82 mRNA/SCR1 RNA and SSA4 mRNA/SCR1 RNA quotients were determined and then normalized to the MED⁺ T = 0 min sample. For both panels, means ± SEM are depicted (N = 3 or 4).

Figure 3

ewe mutations differentially affect RNA Pol II abundance at the heat-shock–induced HSP82 gene. In vivo crosslinking analysis of Pol II at the HSP82 promoter (A), ORF (B), and 3′-UTR (C) either prior to or for the indicated times following an instantaneous 39° heat shock. Pol II abundance at HSP82, quantified using an end-point PCR (gel-based) ChIP assay (Kremer and Gross 2009), was normalized to its abundance at a nontranscribed region (ARS504) evaluated in the same multiplex PCR. For each time point, data are presented for WT and the five congenic ewe mutants in the order indicated in the key. For the WT strain, shown are means ± SEM (N = 3 biological samples; PCR = 12); for rgr1-Δ2 and med10-1002, shown are means ± SEM (N = 4; PCR = 4); and for med7-1002, med21-1002, and med19-1002, shown are means ± SD (N = 2; PCR = 2).

Figure 4

Mutations in the Med14/Rgr1 and Med21 subunits of Mediator diminish the extent of histone displacement at HSP82. Kinetics of histone H3 depletion over the promoter, ORF, and 3′-UTR of HSP82 in WT and mutant cells as assayed by ChIP–qPCR at the indicated times following an instantaneous 30° to 39° temperature shift. H3 globular domain abundance at HSP82 was normalized to its abundance at the PHO5 promoter. This was determined by dividing each HSP82 ChIP value (net nanograms DNA immunoprecipitated) by the corresponding PHO5 ChIP value derived from the same biological sample. Note that H3 occupancy of the PHO5 promoter is independent of both heat-shock and ewe strain background (data not shown and Sekinger and Gross 2001). Depicted are means ± SEM (N = 2; qPCR = 4). Asterisks signify a potentially significant difference between the indicated values (P ≤ 0.06; two-tailed t-test; equal variance). (A–E) H3 occupancy in MED⁺ and the indicated ewe mutant strains. A, C, and E represent one set of immunoprecipitation reactions; B and D represent a second, independently conducted set.

Figure 5

Spontaneous ewe mutants exhibit growth defects on medium containing 6-azauracil. Fivefold serial dilutions of the indicated strains were spotted onto solid media deficient in uracil and containing either no drug (left) or 50 µg/ml 6-AU (right). (Top) Isogenic DST1⁺ and dst1Δ strains; BY4741 strain background. (Middle) HS1001 derivatives (SLY101 strain background) transformed with plasmid pRS316 (CEN-URA3). The deletion strains med16Δ, med5Δ, med20Δ, med12Δ, and cdk8Δ represent the tail, middle, head, and kinase (twice) modules, respectively. (Bottom) HS1004 derivatives (spontaneous suppressors); SLY101 strain background. Plates were incubated for either 3 or 5 days at 30° (left and right, respectively).

Figure 6

Pol II processivity assay reveals a potential role for Med21. Two sets of isogenic strains, derived from either HS1004 (MED⁺ cluster) or BY4741 (DST1⁺ and dst1Δ) and bearing the indicated mutations, were transformed with P_GAL1-PHO5 and P_GAL1-PHO5::lacZ reporter plasmids, and grown in synthetic medium containing 1% galactose and 1% raffinose to log phase. Cells were harvested and Pho5 expression levels were determined by an acid phosphatase assay. The GLAM ratio represents the acid phosphatase activity of cells expressing the long transcript (P_GAL1-PHO5::lacZ) relative to those expressing the short transcript (P_GAL1-PHO5). Depicted are mean values ± SEM of three or six independent transformants of each indicated strain/plasmid combination (N = 3 for med14-1003, med21-1002, med19-1002, cdk8Δ, DST1, and dst1Δ; N = 6 for MED⁺, med7-1002, med10-1002, and med12Δ). *P ≤ 0.05; **P ≤ 0.01.