Stress-Induced MAP Kinase Hog1 Is Part of Transcription Activation Complexes  Paula M. Alepuz, Aleksandra Jovanovic, Vladimir Reiser, Gustav Ammerer  Molecular Cell  Volume 7, Issue 4, Pages 767-777 (April 2001) DOI: 10.1016/S1097-2765(01)00221-0

Figure 1 Hot1 Is a DNA Binding Factor that Directs Hog1 Kinase to the GPD1 Promoter (A) Size of PCR products expected in the ChIP analysis. Location of primer pairs near or at the GPD1 locus on chromosome IV and the position of the GPD1 ORF (arrow) are indicated. (B) Association of Hot1-HA with the GPD1 promoter as measured by ChIPs. Strains PAY106 (wt, left panel), PAY128 (hog1Δ), PAY105 (wt, right panel), PAY136 (msn1), PAY132 (msn2,4), and PAY142 (msn1,2,4) containing an HA epitope–tagged Hot1 were compared before (−) and 5 min after (+) high-osmolarity stress. Control lanes show DNA amplified from extracts without tagged protein (K699, “no tag”) or prior to immunoprecipitation (whole-cell extract, WCE). (C) Hog1-HA binds to GPD1 promoter after osmotstress treatment. ChIP assays on extracts from K4327 (hog1Δ) cells expressing Hog1-HA in the plasmid pVR53. Samples are from unstressed cells or cells exposed for 5 min to 0.4 M NaCl (NaCl), 1.0 M sorbic acid (Sorbic acid), 7.5% ethanol (Ethanol), 39°C (Heat-shock), or 1.5 M sorbitol (Sorbitol). Control lanes are as in (B). (D) Activation and function of Hog1 regulate its binding to GPD1. The pbs2 strain (YVR10) is defective in the MAPKK specific for Hog1; hog1Δ (K4327) carrying the plasmid pVR53-K52S,K53N-HA expresses a tagged Hog1 protein lacking phosphotransfer activity. (E) Hot1 recruits Hog1-HA to the GPD1 promoter. Association of Hog1-HA with GPD1 promoter sequences was measured in different mutant backgrounds. The following strains transformed with pVR53 (Hog1-HA) were assayed by ChIP: K699 (wt), UG43 (hot1), UG44 (msn1), YM24 (msn2,4), and YMR120 (hot1msn1msn2msn4 = ΔTF) Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 2 High Nuclear Concentration of Hog1 Is Insufficient for Chromatin Recruitment (A) In situ detection of Hog1-HA (left panels) and Hog1-NLS-HA (right panels), by immunofluorescence microscopy. Top row of images represent Cy3 signals. Corresponding DAPI images are below. (B) The inability of hog1 strains to grow on elevated salt concentrations is complemented by Hog1-NLS-HA but not by Hog1 K52,K53-HA protein. Transformants were streaked out onto standard agar plates (left) and plates containing 0.4 M NaCl (center) and grown for 2 days at 30°C. The diagram on the right indicates plasmids contained by the hog1 strain (K4327). (C) Hog1 modification as measured on Western blots using antibodies directed against the phosphorylated form (upper panels). The bottom panels present the control with antibodies against the HA tag. Extracts were prepared from unstressed cells (−) and cells under acute stress (+) of the strain hog1Δ (K4327) carrying plasmids pVR53, pVR53-NLS, pVR53-K52S,K53N or YCplac111 (vector). The results with the hot1msn1msn2msn4 strain (YMR120) carrying plasmid Hog1-HA (pVR53) show that the loss of transcription factors does not influence activation of the MAP kinase (lanes labeled ΔTF). Data shown in the left panel are derived from the same exposure. (D) Amplification signals of a GPD1 fragment after chromatin precipitation of Hog1-HA and Hog1-NLS-HA (hog1Δ strain, K4327, carrying the plasmid pVR53 or pVR53-NLS, respectively). Extracts from normally grown cells and osmotically challenged cells are as indicated above. Controls with untagged strain and WCE are also shown. For PCR amplifications, primer pairs were used as in Figure 1 Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 3 Hog1 Is Necessary for Hot1 Association with CTT1 and HSP12 (A) Hog1-HA associates with CTT1 and HSP12 promoters after osmostress. hog1Δ cells expressing Hog1-HA (K4327 strain carrying plasmid pVR53) were exposed to different stress conditions as described in Figure 1C. For each PCR reaction, a mix of three pairs of primers was used, amplifying from −472 to −126 for CTT1 (upper panel) or from −412 to −104 for HSP12 (lower panel). Two more reference primer pairs were included, amplifying from −750 to −420 of CLN2 and from −822 to −567 of CLB2. (B) Association of Hog1-HA with CTT1 and HSP12 in various strains with single or multiple transcription factor deficiencies. ChIPs were made with the strains K699 (wt), UG43 (hot1), UG44 (msn1), YM24 (msn2,4), and YMR120 (hot1msn1msn2msn4 = ΔTF) containing Hog1-HA in the plasmid pVR53, with or without osmostress treatment. (C) Hog1-HA recruitment to CTT1 and HSP12 requires catalytic activity. K4326 strain (hog1Δ) carrying plasmid YCplac111 (no tag), pVR53 (Hog1-HA), or pVR53-K52S,K53N was processed for ChIP assays. (D) Hot1-HA-dependent signals obtained with CTT1 primer pair. Hot1-HA-tagged strains PAY106 (wt) and PAY128 (hog1Δ) and untagged strain (K699) were processed for ChIPs as described above. (E) Hot1-HA binding to HSP12. Strains were as in (C), and right lanes are extracts from hog1Δ strain (PAY128) transformed with plasmid-expressing, catalytically inactive Hog1 (pSWM) Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 4 Interdependent Recruitment of Hog1 and Hot1 at the STL1 Promoter (A) Association of Hot1-HA with STL1 is dependent on Hog1. DNA was isolated from Hot1-HA precipitates derived from wild-type (PAY106) and hog1 (PAY128) cells. Flanking lanes include strain K699 extracts as untagged control (left) and amplifications from whole-cell extract (WCE, right). Primers are supposed to amplify the regions −372 to −113 of STL1, −750 to −420 of CLN2, and −918 to −600 of YCL042w (numbers refer to translational start). The STL1 fragment is indicated. (B) Association of Hog1-HA with STL1 is dependent on Hot1. A wild-type strain (K699) and a hot1Δ strain (UG43) carrying the Hog1-HA-expressing plasmid pVR53 are compared. Controls and primer mixes are as in (A). (C) Activation of Hog1 is necessary for its association with STL1 promoter. Signals for Hot1-HA are compared between wt strain (K699) and pbs2 mutant strain (YVR10) carrying plasmid pVR53. (D) A catalytically inactive Hog1-HA is not recruited to the STL1 promoter. ChIP assays were performed on strain K5327 (hog1Δ) carrying YCplac111 (no tag), pVR53 (Hog1-HA), and pVR53-K52S,K53N (Hog1K52R,K53N-HA) Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 5 Kinetics of Hog1 and Hot1 Promoter Associations (A) Left panels represent ChIP assays performed on a Hog1-HA strain (K4327 carrying pVR53 plasmid), and right panels on a Hot1-HA strain (PAY105). Samples were processed at indicated times after osmostress exposure (0.4 M NaCl) from 0 to 35 min. Samples of the time course are flanked by stressed untagged strain K699 and whole-cell extract controls (WCE). Primer mixtures are as in Figures 1, 4, and 5. (B) Quantifications of ChIP signals represented in (A) using the Quantity One program (Bio-Rad). The numbers correspond to the relative intensity of the positive band with respect to a control band in the same lane that is normally unchanged by osmostress Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 6 Osmostress-Induced Phosphorylation of Hot1 by Hog1 Is Not Essential for Transcription Activation (A) Schematic representation of Hot1 wild-type protein and lexA-Hot1 constructs. Hot1 wild type contains five putative MAP kinase phosphorylation motifs, each containing a serine/proline sequence (positions as indicated). The lexA-Hot1 construct (pPA63) contains a Hot1 fragment encompassing amino acids 140–646, a region containing three of the putative target motifs. In lexA-Hot1m3 (pPA66), all three serines have been mutated to alanines. For details, see Experimental Procedures. (B) Western blot analysis of Hot1 protein under different osmolarity conditions. In the left panel, Hot1-HA from a wild-type background (PAY106, lanes 3–6) was compared to Hot1-HA from a hog1Δ strain (PAY128, lanes 7 and 8). Lanes 1 and 2 show extracts from an untagged control strain (K699). To determine modification by phosphorylation, two samples were treated with λ phosphatase (λ pp, lanes 4 and 6). In the right-hand panel, protein extracts were obtained from cultures of a hot1 strain (UG43) carrying the plasmids pPA54 (Hot1-HA, lanes 9 and 10) and pPA59 (Hot1m3-HA, lanes 11 and 12). All strains either remained unstressed (lanes 1, 3, 4, 7, 9, and 11) or were shocked for 10 min with 0.4 M NaCl (lanes 2, 5, 6, 8, 10, and 12). Monoclonal antibodies against HA epitope were used to detect the Hot1-HA protein. (C) Transcription activation from a lexA-dependent promoter as measured by β-galactosidase activity. Plasmids expressing lexA-Hot1 (pPA63) or lexA-Hot1m3 (pPA66) were cotransformed with plasmid pSH18–18 (containing six copies of lexA operator fused to the reporter gene lacZ) in the strains K699 (wt), UG43 (hot1Δ), PAY164 (rck2Δ), and K4326 (hog1Δ). To check the requirement of the kinase activity of Hog1 to activate transcription, the assay was done with the strain K4326 (hog1Δ) transformed with plasmids pPA63, pSH18–18 and pPA50 (Hog1-K52S,K53N). Strains were grown in selective medium. Cells were stressed by exposure to 0.4 M NaCl for 30 min Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)

Figure 7 Model for Hog1 Functioning as a Transcription Activator Under nonstress conditions, lexA-Hot1 binding to lexA operator activates the transcription machinery at a low level. Inactive Hog1 mainly localizes to the cytoplasm. Upon osmostress, activated Hog1 accumulates in the nucleus, thereby interacting with lexA-Hot1 at the promoter. Besides modifying lexA-Hot1, Hog1 might also engage with and modify one or more components of the transcriptional machinery, such as general transcription factors (GTF) or the RNA polymerase II holoenzyme (Pol II) Molecular Cell 2001 7, 767-777DOI: (10.1016/S1097-2765(01)00221-0)