Mas et al.

Supplemental Figures

Figure S1. Induction of stress genes is affected by mutations on RSC.

RNA levels in wild-type and mutant strains rsc3, rsc8 and rsc9 degron strains (upper panels) or rsc1D and npl6D (lower panels) grown in YPGal medium up to mid-log phase, subjected to 2 h at 37ºC and then subjected to osmotic shock (0.4 M NaCl) for the indicated times are shown. Total RNA was assayed by northern blot for STL1, CTT1 and ACT1 as loading control. Quantification was assessed using the Quantity One program (Bio-Rad).

Figure S2. Hog1 physically associates with the RSC complex.

(A) HA-tagged-Hog1 cells (expressing Hog1 under its own promoter) which contains GST or GST-Rsc9 were subjected to a brief osmotic shock (10 min, 0.4 M NaCl). GST proteins were pulled down by Glutathione Sepharose 4B beads, and the presence of HA proteins was probed by immunoblotting using anti-HA proteins (top). Total extracts represent <20% of total input protein (middle). The amount of precipitated GST proteins was detected using anti-GST (bottom).

(B) Wild type (-) or integrated TAP-tagged-Rsc3 (+) strains were transformed (+) or not (-) with a plasmid carrying Hog1-HA expressed under its own promoter (Hog1-HA) and subjected to a brief osmotic shock (10 min, 0.4 M NaCl). TAP proteins were immunoprecipitated using PAP antibody and the presence of HA proteins was probed by immunoblotting using anti-HA antibodies. Total extracts represent <20% of total input protein. The amount of precipitated TAP protein was detected using anti-PAP.

(C) Hog1 does not coprecipitate with Bdf1. TAP-tagged-Bdf1 strain that express GST or GST-Hog1 was subjected to a brief osmotic shock (10 min, 0.4 M NaCl). GST proteins were pulled down by Glutathione Sepharose 4B beads, and the presence of TAP proteins was probed by immunoblotting using anti-TAP antibody (PAP, Sigma) (top). Total extracts represent <20% of total input protein (middle). The amount of precipitated GST proteins was detected using anti-GST (bottom).

Figure S3. Hog1-dependent histone H3 eviction occurs at osmoresponsive genes but not in ACT1 under stress conditions.

(A) Wild-type and hog1D strains carrying endogenous H3 were grown to mid-log phase and induced with 0.4 M NaCl for 10 min. ChIP was performed to determine binding of H3 (Abcam antibody) to promoter and coding regions of the osmostress response genes CTT1 and GRE2. As internal loading control, PCR samples were amplified with a telomere region (upper band). Control lanes show DNA amplified from extracts prior to immunoprecipitation (Whole-Cell Extract, WCE). Quantification is depicted as fold binding over TEL.

(B) Chromatin as in (A) was also used to determine histone H3 occupancy at the nonstress responsive gene ACT1. Internal and input controls are described in (A), and quantification was assessed as in (A).

Figure S4. Osmostress induces a transient nucleosome eviction.

(A) Histone H3 is transiently evicted from the promoter and ORF of STL1 in a Hog1-dependent manner. Wild type and hog1D strains were grown to mid-log phase and treated with 0.4 M NaCl for the indicated times. Immunoprecipitation against H3 was performed with anti-H3 antibody (Abcam) and binding to STL1 promoter and open-reading frame (ORF) was determined by PCR. As internal loading control, PCR samples were amplified with a telomere region (upper band). Control lanes show DNA amplified from extracts prior to immunoprecipitation (WCE).

Figure S5. Mutations in components of the RSC complex affect cell survival at high osmolarity. Wild-type and the indicated mutant strains were spotted on YPD plates without and with 0.8 M NaCl or 1.2 M NaCl.

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