The SWI/SNF remodeling complex is required for the transcriptional response triggered by cell wall stress. (A) mRNA levels of several CWI-responsive genes were analyzed by RT-qPCR in WT, swi3Δ, and snf2Δ strains after 2 h of CR treatment. Values represent the ratio between CR-treated and nontreated cells. (B) Left, kinetics of MLP1 gene expression analyzed by RT-qPCR in WT and swi3Δ strains at different times of CR treatment. Values represent the ratio between CR-treated and nontreated cells. Right, recruitment of RNA Pol II Rpb1 subunit to MLP1 at MLP1PRO2 region (−143 to +56) determined by ChIP analysis in WT and swi3Δ strains subjected to CR treatment at the times indicated. Results are shown as the fold induction of CR-treated against nontreated samples. (C) The entry of RNA Pol II at MLP1 under cell wall stress determined in WT, rlm1Δ, and slt2Δ strains as indicated in B.

Genome-wide expression profiles of WT (BY4741), swi3Δ, and rlm1Δ strains challenged with CR. (A) Scatter plot of normalized gene expression data in WT and swi3Δ strains. Gene expression ratios (treated/untreated) were plotted against their ORFs using Spotfire software. (B) Heat map obtained by MeV 4.6 software shows gene expression ratios comparing the transcriptional response to CR (treated vs. untreated) in the WT, rlm1Δ, and swi3Δ strains respectively (three columns on the left). Gene expression ratios of a swi3Δ mutant vs. a WT strain in the absence of stress are shown in the fourth column. The genes included in the analysis were those up-regulated in the WT strain by CR treatment. Genes were grouped together (clusters 1–3) on the basis of their dependence on Swi3 for basal and CR-mediated activation (see the text for details). Gene functional categories were assigned according to the information from the Biobase BioKnowledge Library (BKL; http://rous.mit.edu/index.php/Biobase_BioKnowledge_Library). The degree of color saturation represents the expression log₂ ratio value, as indicated by the scale bar.

Deletion of genes that encode SWI/SNF subunits renders cells sensitive to cell wall stress. (A) The indicated strains were spotted on YPD plates without or with 50 μg/ml CR, and plates were incubated for 72 h at 30°C. (B) Zymolyase sensitivity was performed in a 96-well microtiter plate assay with zymolyase 20T, giving concentrations ranging from 125 to 0.12 U/ml YPD. Each well was inoculated with ∼10⁴ cells from an exponentially growing culture. Plates were incubated for 24–48 h at 30°C, and cell growth was determined by measuring absorbance at 550 nm.

Rlm1 occupies the MLP1 promoter in vivo under basal conditions, and it is recruited under cell wall stress in a SWI3- and SLT2-dependent manner. (A) Schematic representation of the MLP1 gene. BOX1 and BOX2 mark the Rlm1-binding sites. Regions amplified by primers for ChIP experiments are shown as horizontal lines. (B) Rlm1-HA association with the MLP1 gene analyzed by ChIP in a WT strain expressing or lacking Rlm1-HA before and after CR addition. The regions of MLP1 analyzed are indicated in the upper left corner. (C) Kinetics of Rlm1-HA recruitment to the MLP1 promoter (MLP1BOX1 region) analyzed by ChIP in WT (left) and swi3Δ mutant (right) strains expressing Rlm1-HA in the absence and presence of CR at the indicated times. (D) Rlm1 recruitment to SRL3 (left) and YLR194C (right) promoters under cell wall stress (CR 30 μg/ml) and nonstress conditions analyzed by ChIP in WT-tagged (Rlm1-HA) and untagged strains, as well as in Rlm1-HA–tagged swi3Δ and slt2Δ strains. Schematic representation of the SRL3 and YLR194C genes, indicating putative Rlm1-binding sites, with the regions analyzed by ChIP (shown as horizontal lines) is included. (E) Rlm1 association to the MLP1BOX1 region of MLP1 analyzed by ChIP in WT and slt2∆ cells expressing Rlm1-HA before and after CR treatment. (F) Rlm1 binding analyzed, as in E, in slt2∆ cells expressing tagged Rlm1-HA transformed with plasmid pRS316-SLT2, pRS316-slt2K54F, or pRS316-slt2T190A/Y192A.

Snf2 and Snf6 are recruited to the activated MLP1 promoter in vivo in a CWI- dependent manner. (A) Association of Snf2 and Snf6 along the different regions of MLP1 (Figure 4A) determined by ChIP in a WT strain grown in the presence or absence of CR. (B) Recruitment of Snf2 at MLP1 (MLP1BOX1), SRL3 (−265 to −135) and YLR194C (−254 to −123) gene promoters analyzed by ChIP as in A in WT, rlm1∆, and slt2∆ strains.

Rlm1 physically interacts with SWI/SNF. (A) Rlm1 associates directly with the SWI/SNF complex in vitro. GST, GST-Rlm1, and GST-Rlm1ΔN were expressed and purified from E. coli, and the SWI/SNF complex was purified from a yeast strain bearing TAP-tag Snf2. SWI/SNF complex was incubated with GST proteins bound to glutathione–Sepharose beads, and interacting proteins were probed using antibodies against GST and Snf2. Specific GST fusion proteins are labeled with an asterisk. Total represents 20% of total input protein. (B) In vivo binding of Rlm1 and SWI/SNF. An rlm1Δ snf2Δ strain was transformed with episomal plasmids expressing Rlm1-Myc and Snf2-HA under their native promoters. Samples were taken from yeast cultures growing in the presence or absence of CR (30 min). Snf2-HA was immunoprecipitated by monoclonal antibodies against HA. Snf2-HA and Rlm1-Myc proteins were detected by Western blotting against HA and Myc epitopes, respectively. Total extract represents 10% of the total input protein.

Lack of Swi3 abrogates chromatin remodeling at MLP1 locus. (A) Kinetics of histone H3 binding to MLP1 promoter (MLP1BOX1 region) in WT and swi3Δ strains under CR treatment at the indicated times. (B) Association of histone H3 to MLP1 promoter (MLP1BOX1) in WT, rlm1Δ, slt2Δ, and snf2Δ strains analyzed by ChIP before and after 90 and 180 min of CR addition. In both cases, results are shown as the ratio between the fold enrichment of the treated against the untreated culture. (C) Nucleosome-scanning analysis of MLP1 locus (from −699 to +161) in WT and swi3Δ strains under basal and cell wall stress conditions. Mononucleosomes were isolated from cultures growing in the absence and presence of CR (3 h). Purified DNA samples were analyzed by qPCR as detailed in Materials and Methods. Bottom, nucleosomes represented as ovals at the indicated position of the MLP1 locus.

Model for transcriptional activation of CWI-responsive genes. In MLP1, those exposed Rlm1-binding sites would allow transcription factor access to the promoter, whereas occluded binding sites within nucleosomes would not be accessible. Under cell wall stress conditions, Slt2 phosphorylates Rlm1, and the SWI/SNF remodeling complex is targeted to the promoters of CWI-responsive genes via interaction with the activated transcription factor. On recruitment, SWI/SNF locally alters nucleosome positioning at the MLP1 promoter, facilitating the binding of Rlm1 to binding sites previously occluded by positioned nucleosomes in a two-step mechanism. Finally, the binding of Pol II stimulates transcription initiation.