Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II

Abstract

In budding yeast, the mitogen-activated protein kinase (MAPK) Hog1 coordinates the transcriptional program required for cell survival upon osmostress. The Hot1 transcription factor acts downstream of the MAPK and regulates a subset of Hog1-responsive genes. In response to high osmolarity, Hot1 targets Hog1 to specific osmostress-responsive promoters. Here, we show that assembly of the general transcription machinery at Hot1-dependent promoters depends on the presence of Hot1 and active Hog1 MAPK. Unexpectedly, recruitment of RNA polymerase (Pol) II complex to target promoters does not depend on the phosphorylation of the Hot1 activator by the MAPK. Hog1 interacts with the RNA Pol II and with general components of the transcription machinery. More over, when tethered to a promoter as a LexA fusion protein, Hog1 activates transcription in a stress- regulated manner. Thus, anchoring of active Hog1 to promoters by the Hot1 activator is essential for recruitment and activation of RNA Pol II. The mammalian p38 also interacts with the RNA Pol II, which might suggest a conserved mechanism for regulation of gene expression by SAPKs among eukaryotic cells.

Fig. 1. Hog1 mediates recruitment of the transcription machinery to stress-responsive promoters in response to stress. Osmostress induces the recruitment of mediator to osmostress-regulated genes as detected by ChIP analysis. Strains containing genomic tags of Rgr1-myc (K9671), Srb10-HA (PAY172), Srb11-HA (PAY257), Kin28-HA (PAY168), TFIIB-myc (K8407) and Rpb1-myc (P156) were grown, and samples for ChIP analyses were taken before (–) and 10 min after (+) the addition of NaCl to a final concentration of 0.4 M. Immuno precipitations were performed using mouse anti-myc or anti-HA monoclonal antibodies. PCR was realized with primers spanning the TATA box of STL1 (arrows) and two pairs of control oligonucleotides spanning the GAL1 and FUS1 gene regions (upper and lower bands, respectively). Control lanes show DNA amplified from extracts without tagged protein (K699, no tag) or prior immunoprecipitation (whole-cell extract, WCE). The same WCE and no tag is presented for experiments carried out in parallel.

Fig. 2. Recruitment of RNA Pol II holoenzyme to promoters depends on specific activators and Hog1 MAPK activity. (A) Hog1 is necessary for TFIIH, TFIIB and Pol II osmotic-stress-dependent association with STL1 and ALD3 promoters. TFIIB-myc strains PAY220 (wt) and PAY217 (hog1Δ), and Kin28-HA strains PAY168 (wt) and PAY173 (hog1Δ) were grown and samples for ChIP analyses were taken as in Figure 1. Pol II binding was detected by using a mouse monoclonal antibody against Rpb1 (8WG16, Covance). Immunoprecipitated samples were processed for ChIPs as described in Materials and methods. Binding to STL1 and/or ALD3 promoters was determined by PCR. (B) Association of Pol II with STL1 and ALD3 promoters requires the presence of specific activators. Cross-linked cell extracts from non-stressed (–) or osmotically stressed (+) wild-type strain (K699) or strains containing a hot1 mutation (UG43) or msn2 msn4 mutations (YM24) were immunoprecipitated using 8WG16 antibody against Pol II. Binding of Pol II to STL1 or ALD3 promoters was assayed as before.

Fig. 3. Hot1 phosphorylation by Hog1 is not required for STL1 activation. (A) Hot1-m5 mutant is not phosphorylated in vitro by Hog1. HA-tagged Hot1 or Hot1-m5 proteins were purified from yeast and incubated with active Hog1 and radioactive ATP (see Materials and methods). Phosphorylated proteins were resolved by SDS–PAGE and transferred to membrane. In vitro phosphorylated proteins were detected by autoradiography (upper panel). HA-tagged Hot1 proteins were detected by immunoblot using anti-HA monoclonal antibodies (lower panel). (B) Hot1 mutant and Hot1 wild type interact with Hog1. Two-hybrid analysis was realized in L40 strain transformed with a LexA-Hog1 plasmid and empty pGAD424 (vector), or containing a wild-type Hot1 (pUG603; Hot1) or an unphosphorylatable Hot1 mutant (pPA89; Hot1-m). β-galactosidase was measured as described in Materials and methods. (C) Hot1-m5 induces STL1 gene expression upon osmostress. Cell cultures of a hot1 mutant strain (PAY181) transformed with the pRS316 plasmid containing Hot1 (pPA97), Hot1-m5 (pPA106) or empty vector were incubated with 0.4 M NaCl at the indicated times. Total RNA was assayed by northern blot analysis for transcript levels of STL1 and RPL28 as a loading control. Quantification data come from the same original blot for each strain and relate to the values at zero time (see Materials and methods).

Fig. 4. Binding of Hot1 to promoters is not sufficient for RNA Pol II recruitment and activation. (A) Promoter-bound Hot1 is not sufficient for initiating Pol II holoenzyme recruitment. Yeast strains were transformed with a centromeric plasmid expressing Hot1-m5 or Hot1-m5- HA at endogenous levels (upper panel), or with multicopy plasmid overexpressing Hot1-HA (left middle panels) or Hot1-m5-HA (right middle panels). ChIPs were performed to determine binding of Hot1, Pol II and TFIIB to STL1 promoter (arrow). The binding of overexpressed wild-type Hot1 (Hot1wt) was analyzed in parallel to overexpressed Hot1-m5 binding to compare the affinity of both proteins with the STL1 promoter (right middle panels). Rpb1-myc (Pol II) binding was analyzed in strains P156 (HOG1) and PAY217 (hog1) (middle panels); Hot1-HA or Hot1-m5-HA binding was analyzed in PAY181(HOG1) and strain PAY218 (hog1Δ). TFIIB-myc binding was analyzed in strains K8407 (wt) and PAY218 transformed with an empty vector or with a plasmid containing a kinase dead Hog1 version (hog1-K/N) (lower panel). (B) Hog1 binds to STL1 promoter upon stress in cells overexpressing Hot1. Binding of Hog1-myc was analyzed by ChIP in the PAY181 (hot1Δ) strain cotransformed with plasmids overexpressing Hot1-HA or Hot1-m5-HA. K699 strain was used as a control (no tag). (C) STL1 expression in cells with constitutively bound Hot1 and Hot1-m5. Wild-type cells with multicopy plasmids overexpressing Hot1 and Hot-m5 were grown in minimal medium and treated with 0.4 M NaCl for 20 min. Total RNA was probed with fragments of STL1 and RPL28A (as a loading control). (D) Hog1 kinase activity is necessary for the initial step of recruitment of the transcription machinery. Rpb1-myc (Pol II) association with the STL1 promoter (arrow) was measured in strains P156 (wt), PAY226 (hot1) and PAY228 transformed with a control vector (hog1) or a plasmid containing the kinase dead Hog1 (hog1-K/N). ChIPs were performed to determine binding to STL1 promoter.

Fig. 5. In vivo and in vitro binding of Hog1 to RNA Pol II holoenzyme. (A) Hog1 physically interacts with the largest subunit of the Pol II in vivo. A myc-tagged Rpb1 strain expressed GST, GST–Hog1 or GST–Hog1DS under the P_TEF1 promoter, or GST–Hog1ΔDS under the P_GAL1 promoter. Cells were grown in the presence of glucose or galactose and samples were taken before (–) or 10 min after (+) treatment with NaCl. GST proteins were pulled down by glutathione– Sepharose 4B and the presence of Rpb1-myc (Pol II) was probed by immunoblotting using anti-myc (upper panel). Total extract represents <20% of total input protein (middle panel). The amount of precipitated GST proteins was detected using anti-GST (lower panel). (B) Hog1 interacts with general components of the transcription machinery. Wild-type strain TM141 was transformed with a plasmid expressing HA-Hog1 under the P_GAL1 promoter and a plasmid expressing GST or a GST-containing protein. Cells were grown in the presence of galactose and samples were taken 10 min after the addition of 0.4 M NaCl. GST proteins were purified as above and HA-Hog1 was detected by western blotting using HA antibodies. Total extracts (middle panel) and GST proteins (lower panel) are shown. (C) Hog1 physically interacts with the largest subunit of the Pol II in vitro. The GST–Hog1 was purified from E.coli and incubated with semipure RNA Pol II holoenzyme. The presence of Rpb1 was probed by immunobloting using 8WG16 antibody against Pol II (upper panel). Total extracts (middle panel) and GST proteins (lower panel) are shown.

Fig. 6. In vivo binding of Hog1 to RNA Pol II is not mediated by specific activators. (A) Hog1 interacts with RNA Pol II in a mutant strain deficient in the transcription factors Hot1, Msn1, Msn2 and Msn4. The YMR120 strain (hot1 msn1 msn2 msn4) was transformed with a plasmid expressing GST, GST–Hog1 or GST–Hog1DS under the P_TEF1 promoter. Cells were grown to mid-log phase and treated with 0.4 M NaCl for 10 min. Coprecipitation experiments were carried out as in Figure 5A. The presence of Rpb1 was probed by immunoblotting by using 8WG16 antibody against Pol II (upper panel). Total extract represents <20% of total input protein (middle panel). The amount of precipitated GST proteins was detected using anti-GST (lower panel). (B) Hot1 does not interact with general components of the transcription machinery. Wild-type strain TM141 was transformed with a plasmid expressing HA-Hot1 (left lanes) or HA-Hog1 (right lanes) and a plasmid expressing GST or a GST-containing protein. Cells were grown and treated with NaCl as before. GST proteins were purified as above and HA-Hot1 or HA-Hog1 was detected by western blotting using antibodies against HA (upper panels). Total extract represents <20% of total input protein (middle panels). The amount of precipitated GST proteins was detected using anti-GST (lower panel).

Fig. 7. Artificially recruited Hog1 stimulates transcription in response to stress in a mediator-dependent manner. (A) Transcription activation from LexA-Hog1 in response to stress. L40 (LexA-lacZ reporter strain) containing hog1Δ or pbs2Δ mutations were transformed with constructs expressing LexA-Hog1, LexA-Hog1DS, LexA-Hog1ΔDS or LexA- Hog1TAYA. β-galactosidase activity was assayed in cells grown to mid-log phase before (open bars) or after (filled bars) a brief osmotic stress (0.4 M NaCl for 30 min). β-galactosidase values are given in nmol/min/mg. To test the degree of LexA-Hog1 phosphorylation, total cell extracts were resolved in SDS–PAGE and immunoblotted with α-LexA antibody (Santa Cruz Biotechnology Inc) to detect LexA-Hog1 (lower panel) and with antiphospho-p38 MAPK antibody (New England BioLabs) to detect phosphorylated Hog1 (upper panel). Data shown are averages of five transformants. (B) Mutations in Srb– mediator genes reduce transcriptional activation by LexA-Hog1. Wild-type L40 or L40 strains containing mutations in srb9, srb10, srb11 or cse2 were transformed with the pBTM116 plasmid expressing LexA-Hog1. β-galactosidase activity was assayed before (open bars) or after (filled bars) a brief osmotic stress as in (A).

Fig. 8. STL1 transcription is upregulated by stress and overexpression of Srb–mediator in a HOG1-dependent manner. (A) Srb–mediator modulates HOG1-mediated STL1 gene expression. Yeast cells containing an integrated STL1::LacZ reporter construct were transformed with a multicopy genomic library and positive clones were selected by their ability to induce STL1. Representative filter β-galactosidase assay demonstrating induction of STL1::LacZ by several positive clones from the screening and their dependence of HOG1 is shown. (B) A wild-type (TM141) and a hog1Δ mutant strain (TM233) carrying an integrated STL1::lacZ reporter construct were transformed with the multicopy plasmid pRS425 either empty (vector) or carrying SRB9, SRB10 or SRB4 genes. Cells were grown and β-galactosidase activity was assayed before (open bars) or after (filled bars) a brief osmotic stress (0.4 M NaCl for 30 min).

Fig. 9. Co-immunoprecipitation of RNA Pol II and p38. HeLa cells were transfected with either pCMV5 or pCMV5-Flag-p38. Transfected cells were serum starved and, when indicated, stimulated with 0.3 M NaCl for 20 min. Lysates from the transfected cells were immunoprecipitated with anti-RNA Pol II antibody (8WG16), and the immunoprecipitates were subjected to immunoblot analysis for flag-p38 using the anti-Flag M2 monoclonal antibody.