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

2003; Springer Nature; Volume: 22; Issue: 10 Linguagem: Inglês

10.1093/emboj/cdg243

1460-2075

Paula Alepúz,

RNA Research and Splicing

Article15 May 2003free access Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II Paula M. Alepuz Paula M. Alepuz Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann-Forschungsstelle, University of Vienna, A-1030 Vienna, Austria Search for more papers by this author Eulàlia de Nadal Eulàlia de Nadal Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Meritxell Zapater Meritxell Zapater Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Gustav Ammerer Corresponding Author Gustav Ammerer Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann-Forschungsstelle, University of Vienna, A-1030 Vienna, Austria Search for more papers by this author Francesc Posas Francesc Posas Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Paula M. Alepuz Paula M. Alepuz Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann-Forschungsstelle, University of Vienna, A-1030 Vienna, Austria Search for more papers by this author Eulàlia de Nadal Eulàlia de Nadal Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Meritxell Zapater Meritxell Zapater Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Gustav Ammerer Corresponding Author Gustav Ammerer Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann-Forschungsstelle, University of Vienna, A-1030 Vienna, Austria Search for more papers by this author Francesc Posas Francesc Posas Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain Search for more papers by this author Author Information Paula M. Alepuz1,2, Eulàlia de Nadal1, Meritxell Zapater1, Gustav Ammerer 2 and Francesc Posas1 1Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain 2Department of Biochemistry and Molecular Cell Biology, Ludwig Boltzmann-Forschungsstelle, University of Vienna, A-1030 Vienna, Austria ‡P.M.Alepuz and E.de Nadal contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2433-2442https://doi.org/10.1093/emboj/cdg243 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Mitogen-activated protein kinase (MAPK) cascades are common signaling modules found in both higher and lower eukaryotic cells (Robinson and Cobb, 1997). Budding yeast has several MAPK cascades, one of which contains a relative of the p38 family of stress-activated MAPKs. This kinase, Hog1, coordinates cellular responses to increases in external osmolarity by inducing diverse osmoadaptive responses (Hohmann, 2002). Recent genome-wide transcriptional studies have revealed that a large number of genes are regulated by osmotic stress in a HOG1-dependent manner, suggesting a key role for the MAPK in stress-induced gene expression (Posas et al., 2000; Rep et al., 2000). There is not a uniform mechanism by which stress-activated MAPKs (SAPKs), and MAPKs in general, modulate gene expression. It has been reported that SAPKs can modify gene regulation by direct phosphorylation of transcription factors, both activators and repressors. In addition, it has been reported that in response to stress, and through intermediate kinases (MSKs), they can induce the phosphorylation of components involved in chromatin remodeling and histones themselves (Kyriakis and Avruch, 2001; de Nadal et al., 2002). In yeast, several transcription factors have been proposed to act under the control of the Hog1 MAPK (i.e. Msn1, Msn2, Msn4, Hot1, Sko1 and Smp1). Each factor seems to be controlling a small subset of the osmoresponsive genes, and thus deletion of a particular transcription factor has a very limited effect on general osmostress gene expression. Although little is known about how Hog1 regulates the activity of downstream factors, two different mechanisms by which it can regulate the activity of a transcription factor have recently been proposed. One mechanism involves the MEF2-like transcription factor Smp1. Smp1 activator is directly phosphorylated by Hog1 on several residues within its transactivation domain, and this phosphorylation by the MAPK is essential for Smp1-mediated gene expression (de Nadal et al., 2003). Similarly, other members of the MEF2 family of transcription factors are regulated by SAPKs (McKinsey et al., 2002). A second mechanism involves the Sko1 transcription factor. Sko1 is an ATF/CREB-related factor (Nehlin et al., 1992; Vincent and Struhl, 1992) which inhibits transcription of several genes that are inducible by osmotic stress (Proft and Serrano, 1999; Garcia-Gimeno and Struhl, 2000). Sko1 represses gene expression by recruiting the general corepressor complex Ssn6–Tup1. Release from Ssn6–Tup1 repression in response to osmotic stress requires direct phosphorylation by the Hog1 MAPK (Proft et al., 2001). Interestingly, Hog1 phosphorylation switches Sko1 activity from a repressing to an activating state, which involves recruiting of SWI/SNF and SAGA complexes (Proft and Struhl, 2002). Here, we analyze the mechanism by which the Hot1 activator controls Hog1-mediated osmostress gene expression. Hot1 is a transcription factor related to Msn1. It controls a small subset of genes involved in the production of osmolyte (Rep et al., 1999). Hot1 interacts with Hog1 (Rep et al., 1999), and this interaction is critical for recruitment of the MAPK to Hot1-dependent promoters and essential for their transcriptional induction upon stress (Alepuz et al., 2001). Thus, apart from the role of Hog1 in the modification of transcription factor activity, its specific association with stress-responsive promoters suggests a new direct involvement of a signaling kinase in complex recruitment and activity. In this work we present evidence for such a model showing that recruitment of the Hog1 MAPK by the Hot1 activator is critical for gene expression. We show that phosphorylation of Hot1 is not required for gene expression or binding of the MAPK to the promoters. Instead the critical step to induce gene expression is the Hog1 directed recruitment of the RNA polymerase (Pol) II complex to the promoter. Our results indicate that Hot1 mediates transcription by the anchoring of a complex that includes active Hog1 MAPK and RNA Pol II holoenzyme. Interestingly, SAPK p38, the mammalian homolog of Hog1, interacts with the core of the RNA Pol II in HeLa cells. These data suggest that a novel conserved mechanism for regulation of gene transcription mediated by stress-activated MAPKs could exist among eukaryotic cells. Results RNA Pol II holoenzyme is recruited to osmoresponsive genes in response to stress Hot1 induces a subset of stress-responsive genes under the control of the Hog1 MAPK in response to osmostress (Rep et al., 1999). Recently, we have shown that the Hot1 activator targets the Hog1 MAPK to osmoresponsive promoters (e.g. STL1) upon stress (Alepuz et al., 2001). However, the underlying mechanism by which Hot1 induces transcription and its regulation by the MAPK is not clearly established. To address this question, we utilized chromatin immunoprecipitation (ChIP) to follow the binding of the transcription machinery to the STL1 promoter before and after osmostress. The STL1 gene is a prototypical Hot1-regulated gene. It is highly expressed in response to osmostress and is completely dependent on the presence of Hot1 and Hog1 MAPK (Posas et al., 2000; Rep et al., 2000). Chromatin from a yeast strain expressing functional epitope-tagged components of the RNA Pol II holoenzyme from their natural locus was immunoprecipitated with antibodies against the HA epitope and analyzed by PCR. We first probed when the Srb–mediator would occupy the STL1 promoter. As shown in Figure 1, Srb10, Srb11 and Rgr1 are found at the STL1 promoter only after osmotic stress. A similar picture was obtained with the core RNA Pol II (Rpb1) and its associated general transcription factors TFIIB (Sua7) and TFIIH (Kin28) (Figure 1). Virtually no signal was detectable in ChIP assays from normal growing cells, whereas stress treatment quickly elicited a strong signal. Thus, the RNA Pol II complex is recruited to the STL1 promoter only in response to stress. Figure 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. Download figure Download PowerPoint Recruitment of the transcription machinery to Hot1-dependent genes depends on active Hog1 and the presence of Hot1 To dissect the role of Hot1 and Hog1 MAPK in the recruitment of the RNA Pol II holoenzyme to osmoresponsive promoters, we analyzed the recruitment of the complex in hog1Δ- and hot1Δ-deficient cells as described above. As shown in Figure 2A, Hog1 is essential for binding of TFIIB and RNA Pol II to the STL1 promoter. Figure 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. Download figure Download PowerPoint The ALD3 gene is strongly responsive to osmostress and depends on Hog1. However, ALD3 expression is not mediated by Hot1 but by the Msn2 and Msn4 transcription factors (Rep et al., 2000). Association of Kin28, TFIIB and Pol II to the ALD3 promoter also correlated with stress induction and Hog1 signaling (Figure 2A). Recruitment of Hog1 to stress-responsive promoters depends on the presence of specific activators. Hot1 is required for binding of Hog1 to STL1, and Msn2/Msn4 are required for Hog1 binding to ALD3 (Alepuz et al., 2001). As revealed by ChIP analysis, binding of Pol II (Rpb1) to STL1 was dependent on the presence of Hot1 and independent of Msn2 and Msn4, whereas binding of Rpb1 to ALD3 was totally dependent on the presence of Msn2 and Msn4 (Figure 2B). The stable recruitment of RNA Pol II holoenzyme seems to correlate closely with the promoter anchorage of Hog1 by specific factors. Therefore, our data suggest that binding of RNA Pol II to osmoresponsive promoters must be a function of both an active Hog1 MAPK and the presence of specific activators. Phosphorylation of Hot1 activator by the MAPK is not required for gene expression The activity of the MAPK was required for Hot1-mediated binding of the RNA Pol II complex to the STL1 promoter, and gene expression upon stress. A possible mechanism of Hot1 regulation is through direct phosphorylation by the MAPK. To test this possibility, we expressed and purified from yeast an HA-tagged wild-type Hot1 and a mutant allele of Hot1 (Hot1-m5) that contains mutations in all putative phosphorylation sites for the MAPK (i.e. Ser30, Ser70, Ser153, Ser360 and Ser410 to Ala). After immunoprecipitation, HA-tagged Hot1 and Hot1-m5 were subjected to an in vitro phosphorylation assay together with active Hog1 (see Materials and methods). As shown in Figure 3A, wild-type Hot1 was phosphorylated by Hog1 whereas the mutated allele Hot1-m5 was not. Figure 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). Download figure Download PowerPoint We then tested whether elimination of the phosphorylation sites affected the binding of Hot1 to the MAPK. Previously, it was shown by two-hybrid analysis that a Gal4 fusion protein containing most of Hot1 was able to interact with Hog1 (Rep et al., 1999). We created a mutant allele of that Gal4 fusion protein with mutations on the phosphorylation sites for the MAPK (Hot1-m). As shown in Figure 3B, two-hybrid analyses indicated that Hot1 was able to interact with Hog1, and binding was not affected by mutation of the Hog1 phosphorylation sites in Hot1. Similar results were obtained with a hog1-K/N mutant (data not shown). To analyze the role of Hog1 phosphorylation in Hot1 transcriptional activity, we transformed a hot1Δ strain with an empty vector or centromeric plasmids carrying wild-type HOT1 or the mutant allele HOT1-m5 expressed under the native HOT1 promoter. Yeast cells were exposed to osmostress, and expression of STL1 was followed by northern blotting. As shown in Figure 3C, expression of STL1 was not induced in a hot1Δ strain carrying the control vector but was strongly induced upon stress in a strain carrying wild-type HOT1. Unexpectedly, both the level of expression and the kinetics of expression of STL1 upon stress in the HOT1-m5 strain were similar to those in the wild-type strain. Thus, these results clearly indicate that phosphorylation of Hot1 by the MAPK is not essential for regulated gene expression upon stress. Artificial binding of Hot1 to promoters is not sufficient for RNA Pol II recruitment and activation There are indications for interdependent promoter binding of Hog1 and Hot1 activator (Alepuz et al., 2001). This situation made it difficult to separate the contribution by the DNA binding factor from that of the kinase in the recruitment of the RNA Pol II. Recently, we observed that an increase in HOT1 dosage will lead to Hog1-independent promoter binding of this factor yet without resulting in any significant elevation of transcription (Figure 4A and C). Moreover, even with Hot1 constitutively present at the STL1 promoter, binding of the mediator, TFIIB and the core RNA Pol II was absolutely dependent on stress and the presence of catalytically competent MAPK (Figure 4A; data not shown). Figure 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. Download figure Download PowerPoint We have shown that the Hot1-m5 allele was unphosphorylatable by Hog1. We then investigated whether binding of Hog1 or RNA Pol II was affected by mutation of the phosphorylation sites in Hot1 in the overexpression system. Binding of Hot1-m5 was similar to that of the wild type when expressed at endogenous levels (Figure 4A, upper panel). Upon overexpression, as for the wild type, binding of Hot1-m5 was constitutive on the STL1 promoter and, more importantly, recruitment of RNA Pol II and binding of Hog1 to the promoter also depended on stress (Figure 4A and B). These results were consistent with the induction of STL1 expression by wild-type Hot1 and mutant Hot1-m5 observed upon stress (Figure 4C). Thus, in a system where binding of Hot1 is unaffected by Hog1, recruitment of RNA Pol II is still dependent on Hog1 activity and independent of the phosphorylation state of the Hot1 activator. To analyze the role of Hog1 kinase activity in the recruitment of RNA Pol II to STL1, we transformed a hog1Δ strain with an empty vector or a vector expressing the catalytically inactive Hog1 enzyme (hog1-K/N). Recruitment of Pol II was completely dependent on Hog1 kinase activity (Figure 4D). Hog1 interacts with the RNA Pol II holoenzyme The Hog1 MAPK regulated Hot1-mediated transcription by a mechanism other than direct phosphorylation of the activator. To gain a better understanding of Hog1-mediated responses we attempted two different approaches: a biochemical identification of direct interactors for Hog1, and a genetic screening to unravel elements required for Hog1-mediated gene expression (described below). It has been reported that interaction of MAPKs with substrates is mediated via conserved docking domains (Sharrocks et al., 2000). Therefore, we fused the C-terminal region of Hog1 (residues 265–435), which contains a hypothetical docking site (DS), to glutathione S-transferase (GST). Cell extracts were prepared from cells carrying GST or GST–Hog1DS, and GST pull-down experiments were carried out with gluthatione–Sepharose beads. Coprecipitating proteins were resolved by SDS–PAGE and silver stained, and prominent bands were analyzed by high-accuracy peptide mass mapping using MALDI analysis (described in Materials and methods). Two of the most abundant proteins present in the coprecipitation with Hog1DS corresponded to the α and β subunits of the RNA polymerase (Rpb1 and Rpb2). Binding of Hog1 to Rpb1 was confirmed by direct coprecipitation experiments. Yeast cells containing a chromosomally myc-tagged Rpb1 were transformed with plasmids that expressed GST-tagged full-length Hog1, Hog1DS (residues 265–435) or Hog1ΔDS (residues 1–301, corresponding to the kinase domain). As shown in Figure 5A, Rpb1 coprecipitated with full-length Hog1 and with the C-terminal region (Hog1DS) but not with Hog1ΔDS. Whereas binding of Rpb1 to the full-length Hog1 was stress dependent, binding to Hog1DS was constitutive. Thus, binding of Rpb1 to Hog1 is mediated by the non-catalytic region that comprises the MAPK docking site. To assess whether binding of Hog1 with the core of the RNA Pol II was direct, we performed an in vitro binding assay using purified Hog1 from Escherichia coli with Rpb1-myc from yeast (described in Materials and methods). As shown in Figure 5C, Hog1 was able to interact with purified Rpb1. Figure 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 PTEF1 promoter, or GST–Hog1ΔDS under the PGAL1 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 PGAL1 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. Download figure Download PowerPoint We next tested whether Hog1 interactions extended to other components of the transcription initiation complex. One of the general factors closely associated with the activation of the core polymerase is TFIIH. Wild-type cells expressing an HA-Hog1 protein were transformed with a plasmid expressing GST-tagged Kin28, the catalytically important subunit of TFIIH. A GST pull-down from osmotically challenged yeast cell extracts showed that Hog1 is also closely associated with this general factor (Figure 5B), and supported the notion that Hog1 interactions might take place with larger entities and not with the individual components. We undertook similar GST pull-down assays with parts of the Srb–mediator that identify different subcomplexes of the holoenzyme (Woychik and Hampsey, 2002). The results (Figure 5B) provided strong evidence that Hog1 can indeed interact with certain forms of the RNA polymerase holoenzyme. One exception was provided by Cse2/Med9, a component of one of the proposed Rgr1 mediator modules, documenting a certain level of selectivity in the observed interactions between the MAPK and the RNA Pol II holoenzyme. Binding of the Hog1 MAPK to the RNA Pol II holoenzyme is unlikely to be mediated by specific activators, because this interaction was not affected in a strain deficient in msn1Δ, msn2Δ, msn4Δ and hot1Δ (Figure 6A). Furthermore, binding of Hot1 to components of the RNA Pol II holoenzyme was assayed in parallel to Hog1. As shown in Figure 6B, Hog1 was able to interact with Kin28 and Sin4, whereas Hot1 was unable to interact with these components. Thus, our data suggest that the recruitment of RNA Pol II holoenzyme to Hot1-dependent promoters is carried out by Hog1 and not by Hot1, further indicating that Hog1 acts as an adaptor for Hot1 in the assembly of the RNA Pol II complexes at the STL1 promoter. Figure 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 PTEF1 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 10-fold under osmostress conditions (Figure 7A). The difference in transcription between normal and stressed cells seemed to depend on Hog1 phosphorylation, because it was lost in cells deficient in the MAPKK responsible for Hog1 phosphorylation and activation, i.e. pbs2Δ cells (see inset). It is also worth noting that the complete active Hog1 protein was required for transcriptional activity, because neither the Hog1DS nor the Hog1ΔDS proteins fused to a LexA were able to activate transcription.