FIGURE 2.

Mutations in Tail subunits Med14, Med15, and Med16 diminish Hsf1 occupancy of heat shock-induced promoters, whereas mutations in the Mediator Head, Middle, and Kinase modules have no effect. A, Hsf1 ChIP analysis of HSP promoters under non-inducing and 15 min heat shock-inducing conditions in isogenic MED⁺ and med14/rgr1-Δ2 strains DY150 and DY2694. The assay was conducted as in Fig. 1 except that an Hsf1-specific polyclonal antibody was used; preimmune signal was subtracted from each ChIP signal. The abundance of Hsf1 in response to heat shock was quantified relative to its abundance at each promoter under non-heat shock conditions (T = 0 min). Depicted are means ± S.D. (error bars); n ≥ 3. B, dynamic range of Hsf1 occupancy at HSP promoters in MED⁺ and rgr1-Δ2 strains during the first 15 min of heat shock (data from A). C, Hsf1 occupancy of HSP promoters in isogenic MED⁺ and med15Δ cells (BY4741 background) either maintained at 30 °C (0 min) or subjected to a 39 °C heat shock for the indicated times. Shown are means ± S.D., n = 3. D, as in C, except isogenic MED⁺ and med16Δ cells were examined. E and F, as in C, except Hsf1 occupancy of SSA4 and HSP82 promoters was assayed in MED⁺ and isogenic med19-1001, med10-1001, med21-1002, and cdk8Δ strains. Asterisks signify a significant difference in Hsf1 occupancy between WT and mutant (p < 0.05; two-tailed t test; equal variance).