The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment
2019; Springer Nature; Volume: 38; Issue: 24 Linguagem: Inglês
10.15252/embj.2019102566
ISSN1460-2075
AutoresRyosuke Takii, Mitsuaki Fujimoto, Masaki Matsumoto, Pratibha Srivastava, Arpit Katiyar, Keiichi I. Nakayama, Akira Nakai,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle28 October 2019free access Transparent process The pericentromeric protein shugoshin 2 cooperates with HSF1 in heat shock response and RNA Pol II recruitment Ryosuke Takii orcid.org/0000-0001-7646-4029 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Mitsuaki Fujimoto Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Masaki Matsumoto Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Pratibha Srivastava orcid.org/0000-0001-8563-7526 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Arpit Katiyar Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Keiich I Nakayama Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Akira Nakai Corresponding Author [email protected] orcid.org/0000-0002-1833-7061 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Ryosuke Takii orcid.org/0000-0001-7646-4029 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Mitsuaki Fujimoto Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Masaki Matsumoto Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Pratibha Srivastava orcid.org/0000-0001-8563-7526 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Arpit Katiyar Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Keiich I Nakayama Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Akira Nakai Corresponding Author [email protected] orcid.org/0000-0002-1833-7061 Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Author Information Ryosuke Takii1, Mitsuaki Fujimoto1, Masaki Matsumoto2, Pratibha Srivastava1, Arpit Katiyar1, Keiich I Nakayama2,3 and Akira Nakai *,1 1Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan 2Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 3Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan *Corresponding author. Tel: +81 836 22 2214; E-mail: [email protected] EMBO J (2019)38:e102566https://doi.org/10.15252/embj.2019102566 Correction added on 4 November 2019, after first online publication: HSF1 was replaced with SGO2. PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter-bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress-specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO211 Correction added on 4 November 2019, after first online publication: HSF1 was replaced with SGO2. to the HSP70 promoter. SGO2-mediated binding and recruitment of Pol II with a hypophosphorylated C-terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1-SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress-specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response. Synopsis HSF1 mediates stress-specific RNA Polymerase II (Pol II) recruitment to heat shock genes via partly understood mechanisms. Here, comparative substitution and deletion analyses of HSF1 vertebrate paralogs highlight pericentromeric adapter protein shugoshin 2 (SGO2), better known for its roles in chromosome segregation, as unexpected HSF1 co-factor in chaperone gene expression. Anole lizard HSF1 residues Pro17 and Ser326 are required for HSP70 induction in mouse embryonic fibroblasts. SGO2 interacts with HSF1 in Ser326-phosphorylation-dependent manner. SGO2 interacts with Pol II via its hypophosphorylated C-terminal domain, and facilitates Pol II recruitment to the HSP70 promoter. Impaired HSF1-SGO2 interaction impairs proteostatic capacity Introduction Transcription by RNA polymerase II (Pol II) is regulated at multiple processes including formation of the preinitiation complex (PIC), initiation, pausing, and elongation. The formation of the PIC containing Pol II and general transcription factors (GTFs), which include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, at gene promoters is one of the major regulatory processes in eukaryotic gene expression. This process is facilitated by transcriptional activators that bind to specific DNA sequences near target genes and recruit coactivators including chromatin remodeling complexes and histone-modifying enzymes, which open the structure of chromatin through regulating its post-translational modifications (Fuda et al, 2009; Sainsbury et al, 2015; Venkatesh & Workman, 2015). Coactivators, such as Mediator, also interact directly with components of the transcription machinery and hence facilitate the recruitment of Pol II (Hantsche & Cramer, 2017; Soutourina, 2018), although some activators can directly interact with and stabilize them. The activator-dependent recruitment of the PIC including Pol II is highly regulated, especially in inducible genes, the expression of which is rapidly and robustly upregulated in response to external stimuli (Weake & Workman, 2010). The heat shock response (HSR) is an adaptive mechanism against proteotoxic stresses that adjust proteostasis capacity primarily via the induction of heat shock proteins (HSPs), or chaperones, which facilitate protein folding (Lindquist, 1986; Balch et al, 2008; Morimoto, 2008). The HSR is mainly regulated at the level of transcription by evolutionarily conserved heat shock transcription factors (HSFs) that bind to HSR elements (HSEs) in eukaryotes (Wu, 1995; Morimoto, 1998; Nakai, 2016; Gomez-Pastor et al, 2018). In contrast to the single HSF gene in yeast, worm, and fruit fly, four HSF genes (HSF1–HSF4) exist in vertebrates. Among these, HSF1 regulates HSP expression during heat shock in mammals and lizards, whereas it is dispensable in avians, in which HSF3 takes the place of HSF1 (Fujimoto & Nakai, 2010; Takii et al, 2017). HSF1 mostly remains as an inert monomer in unstressed conditions. In response to heat shock, it is converted to an active trimer that binds to HSEs, and its transcriptional activity is enhanced by post-translational modifications including phosphorylation (Björk & Sistonen, 2010; Nakai, 2016; Gomez-Pastor et al, 2018). HSF1-mediated induction of HSPs as well as non-HSPs is associated with elevated proteostasis capacity and cell survival. Transcriptional mechanisms of the HSR have been extensively studied. In Drosophila, GAGA-associated factor constitutively binds to the HSP70 promoter, which allows for the establishment of an open chromatin environment and paused Pol II at the promoter-proximal region (Jonkers & Lis, 2015; Duarte et al, 2016). In response to heat shock, trimerized HSF quickly occupies HSEs in the promoter (Guertin & Lis, 2010). Promoter-bound HSF directly recruits Mediator (Park et al, 2001) and TATA box-binding protein, a core component of TFIID (Mason & Lis, 1997), which facilitate PIC formation and recruitment of Pol II. It also recruits other coactivators, including P-TEFb and TIP60, that promote rapid loss of nucleosomes and the release of stalled Pol II, which is another highly regulated process (Jonkers & Lis, 2015). In mammals, constitutively bound HSF1 in complex with replication protein A (RPA) promotes the establishment of paused Pol II and an open chromatin environment (Fujimoto et al, 2012). It also recruits PARP13-PARP1 complex to prepare for the response (Fujimoto et al, 2018). During heat shock, activated HSF1 heavily occupies HSEs in the HSP70 promoter and dramatically induces its transcription (Vihervaara et al, 2013; Takii et al, 2015; Mahat et al, 2016), by recruiting various kinds of coactivators including ASC-2 (Hong et al, 2004), MLL1 (Chen et al, 2014), and BRG1 (Sullivan et al, 2001; Corey et al, 2003), and by causing the redistribution of PARP1 (Fujimoto et al, 2018). These coactivators promote establishment of an active chromatin state and may thereby facilitate PIC formation during heat shock (Corey et al, 2003; Takii et al, 2015; Fujimoto et al, 2018). However, the molecular mechanisms by which the stress-inducible activator HSF1 directly facilitates PIC formation and the recruitment of Pol II are not well known in mammalian cells. Here, we generated HSF1 mutants lacking transcriptional activity using an evolutionary approach and identified activity-dependent components of HSF1 complexes bound to the HSP70 promoter in vitro in mouse cell extracts. We revealed that the expression of HSPs during heat shock is markedly enhanced by a component of the HSF1 transcription complexes, shugoshin 2 (SGO2), which regulates chromosome segregation during meiosis in mice (Watanabe, 2005; Gutiérrez-Caballero et al, 2012; Marston, 2015). Remarkably, HSF1-SGO2 facilitated PIC formation in the HSR by directly recruiting Pol II to promoters. Furthermore, the HSF1-SGO2 complex maintained proteostasis capacity in heat shock conditions. Results AsHSF1 mutants lacking transcriptional activity It is important to identify HSF1 mutants with different potential to activate HSP genes in order to uncover coregulators of HSF1. To this end, we used an evolutionary approach involving comparative analysis of HSF1 paralogs in vertebrates. We confirmed that the overexpression of anole lizard (Anolis sagrei) HSF1 (AsHSF1) as well as human HSF1 (hHSF1) in HSF1-null mouse embryonic fibroblasts (MEF) restored the heat shock-induced expression of HSP70 mRNA, whereas that of chicken HSF1 (cHSF1) did not (Appendix Fig S1A) (Takii et al, 2017). We investigated the ability of various cHSF1 mutants, parts of which were swapped with corresponding AsHSF1 regions, to induce HSP70 mRNA in this experimental system. It was revealed that substitution of the cHSF1 DNA-binding domain (DBD) with that of AsHSF1 (cHSF1-ch2 and -ch3) moderately restored the induction of HSP70 mRNA during heat shock, and further substitution of region X (cHSF1-ch4) fully restored the induction (Appendix Fig S1B). Furthermore, substitution of only Ser21 in cHSF1 DBD with the corresponding Pro17 in AsHSF1 DBD (cHSF1-S21P) modestly restored the induction (Fig 1A and Appendix Fig S1C and D). This proline residue is located at the N-terminal end of the DBD and is conserved in eukaryotic HSFs except cHSF1 (Nakai & Morimoto, 1993) (Appendix Fig S1E). Moreover, the 24 amino acids containing the evolutionarily conserved site d or f (called the d-region) in region X are deleted in cHSF1 (Nakai & Morimoto, 1993; Fujimoto et al, 2010) (Appendix Fig S1F), and insertion of the AsHSF1 d-region (cHSF1-S21P/ins-d) restored the full induction (Fig 1A and Appendix Fig S1G). In contrast, substitution of Pro17 in AsHSF1 with serine and deletion of the d-region containing 24 amino acids (AsHSF1-P17S/Δd) reduced the induction of HSP70 mRNA (Fig 1B). hHSF1 mutants possessing the same mutations (hHSF1-P16S/Δd) also have reduced potential to induce HSP70 mRNA, although the reduction level was modest (Fig 1C). Figure 1. AsHSF1 mutants lacking transcriptional activity in mouse cells Potential of cHSF1 mutants to induce HSP70 expression. HSF1−/− MEF cells were infected with adenovirus expressing wild-type cHSF1, cHSF1-S21P, cHSF1-ins-d (d-region of AsHSF1 inserted), cHSF1-S21P/ins-d (left), or GFP as a control. These HSF1+/+ cells were untreated (Cont.) or treated with heat shock at 42°C for 30 min (HS), and HSP70 mRNA levels were quantified by RT–qPCR. The levels relative to that in control Ad-GFP-infected HSF1+/+ cells were shown (middle). Extracts from cells before heat shock were subjected to immunoblotting using anti-HSF1 or anti-β-actin antibody (right). Potential of AsHSF1 mutants to induce HSP70 expression. Cells were infected with adenovirus expressing wild-type AsHSF1, AsHSF1-P17S, AsHSF1Δd, or AsHSF1-P17S/Δd (left). HSP70 mRNA levels were quantified by RT–qPCR before and after heat shock (middle). Extracts were subjected to immunoblotting (right). Potential of hHSF1 mutants to induce HSP70 expression. Cells were infected with adenovirus expressing wild-type hHSF1, hHSF1-P16S, hHSF1Δd, or hHSF1-P16S/Δd (left). HSP70 mRNA levels were quantified by RT–qPCR (middle), and extracts were subjected to immunoblotting (right). Alanine substitution of serine and threonine in the AsHSF1 d-region. Serine and threonine were substituted with alanine (red) in the N-terminal half of the d-region, which was required to induce HSP70 expression (left; Appendix Fig S1H). Cells were infected with adenovirus expressing wild-type and mutated AsHSF1 (upper right), and HSP70 mRNA levels were quantified by RT–qPCR before and after heat shock (lower right). Amino acids corresponding to AsHSF1-S326 are highlighted in blue. Data information: Error bars, SD (n = 3). Asterisks indicate ***P < 0.001 by Student's t-test in (A–D). Download figure Download PowerPoint We next searched for amino acids in the AsHSF1 d-region required for transcriptional activity and found that deletion of the N-terminal 12 amino acids (AsHSF1-del4), but not other amino acids, partially reduced HSP70 expression (Appendix Fig S1H). This short region contains 4 serine or threonine residues, one of which (AsHSF1-Ser326) corresponds to hHSF1-Ser326, whose phosphorylation is accompanied by transcriptional activation (Guettouche et al, 2005). It turned out that a mutation of AsHSF1-Ser326 is sufficient for reduced HSP70 expression during heat shock (Fig 1D). In conclusion, our evolutionary approach uncovered amino acid residues of AsHSF1, Pro17 and the d-region including Ser326, which are required to induce HSP70 expression in mouse cells. Identification of coregulators recruited to AsHSF1 complexes in the HSP70 promoter We examined the in vivo binding of ectopically expressed AsHSF1 mutants (AsHSF1-P17S, AsHSF1Δd, and AsHSF1-P17S/Δd) to the HSP70 promoter in HSF1-null MEF cells and found that their occupancy was elevated dramatically during heat shock like that of wild-type AsHSF1 (Appendix Fig S2A and B). In contrast, Pol II occupancy on the pausing and coding regions was enhanced at lower levels during heat shock in cells expressing AsHSF1 mutants than that in cells expressing wild-type AsHSF1 (Appendix Fig S2C). Consistently, histone H3 occupancy was higher in the same cells. These results suggested that AsHSF1 mutants cannot recruit coregulators, which enhance Pol II occupancy, to the HSP70 promoter during heat shock. To identify coregulators of AsHSF1, we analyzed complexes bound in vitro to AsHSF1 and three AsHSF1 mutants on HSP70 promoter using a biotin-labeled DNA pull-down assay (Foulds et al, 2013) (Fig 2A). A human HSPA1A (HSP70-1) promoter fragment (wt70P) containing the proximal and distal HSE sequences (pHSE and dHSE) or its HSE-mutated promoter fragment (mu70P; Appendix Fig S2D) was incubated with a nuclear extract of MEF cells infected with each adenovirus expression vector, and bead-bound proteins were resolved by SDS–PAGE (Appendix Fig S2E) and identified by mass spectrometry (MS). A total of 1,870 proteins were identified in AsHSF1:wt70P complex, and identified peptide numbers of the 676 proteins were higher than those in AsHSF1:mu70P complex (difference of peptide numbers > 1; HSE-dependent components; Fig 2B and C). This assay successfully identified already known coregulators of HSF1, such as p300, CBP, ATF1, CREB (Takii et al, 2015), GCN5 (Zelin et al, 2012), TRIM28 (Bunch et al, 2014), CHD4 (Khaleque et al, 2008), STUB/CHIP (Dai et al, 2003), and a set of Mediator (Kim & Gross, 2013) as well as HSF2 and HSF4 (Ostling et al, 2007; Fujimoto et al, 2008), in addition to many proteins with various functions (Fig 2C). Among HSE-dependent components, we further selected 179 proteins whose differences in peptide numbers between AsHSF1:wt70P and AsHSF1:mu70P complexes are higher than those between AsHSF1-P17S/Δd:wt70P and AsHSF1-P17S/Δd:mu70P complexes (difference of peptide numbers > 1). Numbers of identified peptides of these proteins, including p300, CBP, STUB/CHIP, CHD4, and TRIM28, were correlated with the transcriptional activity of AsHSF1 (activity-dependent components; Fig 2D and Table EV1). Figure 2. Identification of coregulators recruited to AsHSF1 complexes on the HSP70 promoter Schematics of the in vitro assay for the detection of AsHSF1 complexes on the HSP70 promoter. The human HSPA1A (HSP70-1) promoter fragment (wt70P, −448 to +13) containing pHSE and dHSE or its HSE-mutated promoter fragment (mu70P) was immobilized on magnetic beads and incubated with nuclear extract of heat-shocked HSF1−/− MEF cells expressing AsHSF1 or its mutant. Bead-bound proteins were resolved by SDS–PAGE and identified by MS. Numbers of HSE-dependent, activity-dependent, or activity-independent components of AsHSF1 complexes. The HSE-dependent 676 components consist of proteins, whose peptide numbers identified in AsHSF1:wt70P complex were higher than those in AsHSF1:mu70P complex (difference of peptide numbers > 1; a full list is shown in Table EV1). The activity-dependent 179 components consisted of proteins, whose differences in peptide numbers between AsHSF1:wt70P and AsHSF1:mu70P complexes are higher than those between AsHSF1-P17S/Δd:wt70P and AsHSF1-P17S/Δd:mu70P complexes (difference of peptide numbers > 1). HSE-dependent components of AsHSF1 complexes on the HSP70 promoter. Among 676 HSE-dependent components shown in (B), 85 transcription-related and 115 other proteins, whose difference of peptide numbers was more than three, are classified (difference of peptide numbers > 3). Gray bars indicate known coregulators of HSF1. HSE- and activity-dependent components of AsHSF1 complexes on HSP70 promoter. Major 21 proteins out of 179 HSE- and activity-dependent components are listed (difference of peptide numbers > 5). Numbers of identified peptides from AsHSF1:wt70P and AsHSF1:mu70P complexes and their differences are shown (left). Relative enrichment is indicated as a heatmap with color scale in each complex (right). Download figure Download PowerPoint SGO2 enhances HSP70 expression during heat shock We examined the impact of the HSE- and activity-dependent components on the HSR in mouse cells and found that knockdown of five components including MED12, SGO2, PHF6, MCCC1, and MORF4L2, out of 10 newly identified components, significantly reduced the expression of HSP70 mRNA during heat shock (Fig 3A and Appendix Fig S3A). Knockdown of the other five components enhanced the induced HSP70 expression or had no effect like that of p300 (Takii et al, 2015). Among them, we analyzed the roles of SGO2, which regulates chromosome segregation during meiosis in mice (Llano et al, 2008; Orth et al, 2011). We found that SGO2 protein was substantially expressed in interphase cells including cells arrested at G1/S phase of the cell cycle (Appendix Fig S3B). Proportions of G1 and G2/M were nearly constant in cells infected with Ad-sh-mSGO2 for 3 days (Appendix Fig S3B), excluding a possibility that the reduced HSP70 expression was due to aberrant cell cycle progression. Figure 3. SGO2 enhances HSP70 expression during heat shock Functional screening of major components in AsHSF1 complexes. We selected 10 proteins out of major 21 HSE- and activity-dependent components (Fig 2D), by removing known coregulators, an enzyme, and some translation-related proteins. MEF cells were infected with adenovirus expressing shRNAs for genes of these HSE- and activity-dependent components, and treated with heat shock at 42°C for 30 min. HSP70 mRNA levels were quantified by RT–qPCR, and induction levels relative to that in SCR-treated cells (%) are shown. Stability of SGO2 during heat shock. MEF cells were treated with heat shock at 42°C for the indicated periods. Nuclear and cytoplasmic extracts from these cells were prepared and subjected to immunoblotting of SGO2 and HSF1, as well as a nuclear protein lamin B1 and HSP90, which localizes predominantly in the cytoplasm. SGO2 localized in the nucleus, and HSF1 accumulated in the nucleus during heat shock. A star indicates non-specific bands. Expression of HSP mRNAs in SGO2-knockdown cells during heat shock. Cells were infected for 72 h with adenovirus expressing shRNA for SGO2 (KD1 or KD2) or scrambled RNA (SCR), and treated with heat shock at 42°C for 20, 40, and 60 min. mRNA levels of HSP70 (HSPA1A/B), HSP40 (DNAJB1), and HSP25 (HSPB1) were quantified by RT–qPCR, and the levels relative to that in control SCR-treated cells (fold induction) are shown (upper left). HSP70 mRNA levels in control conditions are shown separately (upper right). Cell extracts were prepared using NP40-lysis buffer, and subjected to immunoprecipitation and immunoblotting using antibody for SGO2 or SGO1 (lower). Expression of HSPs in SGO2-knockdown cells during heat shock. SGO2-knockdown cells were treated with heat shock at 42°C for 30 min and recovered for the indicated periods (HS + Rec.). Cell extracts were prepared using NP40-lysis buffer and subjected to immunoblotting. Expression of HSP70 mRNAs during treatment with proteotoxic stress inducers. SGO2-knockdown cells were treated with heat shock at 42°C for 30 min (HS), 10 μM MG132 for 3 h, 5 mM AZC for 3 h, and 50 μm sodium arsenite for 6 h (As). HSP70 mRNA levels were quantified by RT–qPCR. Expression of HSP70 protein in HSF1−/− MEF cells stably expressing AsHSF1. SGO2 was knocked down in MEF-AsHSF1 cells (clone #9). These cells were untreated (Cont.) or treated with heat shock at 42°C for 30 min and then recovered for 6 h (HS). Cell extracts were prepared and subjected to immunoblotting. Ectopically expressed AsHSF1 is stabilized during heat shock (Takii et al, 2017). Data information: Error bars indicate SD (n = 3). Asterisks indicate *P < 0.05 or **P < 0.01 by ANOVA in (C) and by Student's t-test in (E). Download figure Download PowerPoint Shugoshin 2 was stable in the nucleus during heat shock at 42°C for 5 and 10 min, although it decreased thereafter (Fig 3B). We found that knockdown of SGO2 reduced mRNA levels of HSP70, HSP40, and HSP25 at 20, 40, and 60 min after heat shock (Fig 3C). To estimate accumulation of HSPs, we also examined protein levels during recovery for 1, 3, or 6 h after heat shock at 42°C for 30 min, and showed reduction of HSP levels in SGO2-knockdown cells (Fig 3D). The basal expression of HSP mRNAs was unaffected by SGO2 knockdown (Fig 3C). We confirmed the reduced HSP70 expression in other mouse cell lines including C2C12, F9, and Neuro-2a cells (Appendix Fig S3C). Furthermore, the induction of HSP70 mRNA during treatment with other proteotoxic stress inducers, including a proteasome inhibitor MG132, a proline analog l-azetidine-2-carboxylic acid (AZC), and sodium arsenite (As), was also reduced by SGO2 knockdown (Fig 3E). To investigate whether the transcriptional role of SGO2 is specific to proteotoxic stress, we examined the expression of inducible genes in response to DNA damage and elevation of cAMP level. It was revealed that forskolin-induced expression of AREG and CXCL1 mRNAs and doxorubicin-induced expression of LHX15 and RAP2B mRNAs were unaffected by SGO2 knockdown (Fig EV1). Knockdown of SGO1, another member of the shugoshin family, did not alter the expression of HSP70 mRNA during heat shock (Appendix Fig S3D). These results indicated that mouse SGO2 is not generally required for transcription, but distinctively enhances the expression of HSPs during proteotoxic stresses. Click here to expand this figure. Figure EV1. Effect of SGO2 knockdown on expression of inducible genes by other stimuliMEF cells were infected for 72 h with adenovirus expressing shRNA for SGO2 (SGO2-KD1 or SGO2-KD2), HSF1, p300, CBP, or scrambled RNA (SCR). Amphiregulin (AREG) and CXCL1 mRNA levels during treatment with forskolin (20 μM) for 1 h were quantified by RT–qPCR, and the levels relative to those in control SCR-treated cells are shown (left upper). The forskolin-mediated induction was reduced by p300 or CBP knockdown (Takii et al, 2015), but not by SGO2 or HSF1 knockdown. The mRNA levels of LHX15 and RAP2B, HSF1-independent doxorubicin-inducible gene products (Fujimoto et al, 2018), during the treatment with doxorubicin (0.5 μM) for 24 h were also quantified by RT–qPCR (left lower). Cell extracts were prepared using NP40-lysis buffer and subjected to immunoblotting (right). Error bars, SD (n = 3). Asterisks indicate *P < 0.05 or **P < 0.01 by Student's t-test. An arrowhead indicates SGO2 band, and an asterisk indicates non-specific band in SGO2 blot. Download figure Download PowerPoint A lizard HSF1 mutant, AsHSF1-P17S/Δd, had little potential to induce HSP70 mRNA during heat shock, whereas a human HSF1 mutant, hHSF1-P16S/Δd, had a reduced but measurable potential (Fig 1B and C). This observation led us to examine the effects of SGO2 knockdown on HSP70 expression in MEF cells expressing AsHSF1. The protein levels of HSP70, HSP40, and HSP25 were induced dramatically in these cells during recovery for 6 h after heat shock. Surprisingly, they were hardly induced by SGO2 knockdown (Fig 3F). This result further supported the assertion that mouse SGO2 plays an important role in the HSR. Phosphorylated HSF1 at Ser326 recruits SGO2 to HSP70 promoter We confirmed that HSF1 was co-precipitated with SGO2 from an extract of heat-shocked cells, but not from that of unstressed cells (Fig 4A). Therefore, we performed ChIP assays using primer sets for the mouse HSPA1A (HSP70.3) locus (Fig 4B). HSF1 constitutively bound to pHSE in the HSPA1A promoter (we refer to this as the HSP70 promoter later) at a low level, but not to dHSE, and its binding dramatically increased at both pHSE and dHSE during heat shock (Fig 4C) (Takii et al, 2015). SGO2 did not occupy any region within the HSP70 locus in unstressed condition. Remarkably, SGO2 occupied pHSE, but not dHSE, at its highest level at 5 min after heat shock, and its occupancy decreased slightly afterward (Fig 4C). Simultaneously, SGO2 moderately occupied the downstream pausing region, where Pol II is stalled. HSF1 knockdown abolished the heat shock-induced occupancy of SGO2 on the pHSE and pausing region, whereas SGO2 knockdown slightly reduced HSF1 binding at 5 min after heat shock and had no effect at 30 min (Fig 4D and Appendix Fig S4A). SGO2 also occupied the HSP40 and HSP25 promoters during heat shock, and its occupancy was blocked by HSF1 knockdown (Fig 4D). These results indicated that HSF1 recruits SGO2 to HSP promoters during heat shock. A further implication is that HSF1 may recruit different coactivators depending on the location of its binding site. Figure 4. Phosphorylated HSF1 at Ser326 recruits SGO2 to the HSP70 promoter Interaction of HSF1 with SGO2. Extracts in NP-40 lysis buffer were prepared from control MEF cells (C) and cells treated with heat shock at 42°C for 30 min (HS). Complexes co-immunoprecipitated using SGO2 antibody were blotted with HSF1 or SGO2 antibody. An arrowhead indicates SGO2 band. Schematic view of the mouse HSPA1A locus. Shaded boxes indicate DNA regions (1–9) amplified by RT–qPCR. Occupancy of HSF1 and SGO2 on the HSPA1A locus in control MEF cells (Cont.) and cells treated with heat shock at 42°C for 5, 10, and 30 min (HS). ChIP-qPCR on each region shown in (B) was performed. HSF1-dependent recruitment of SGO2 to HSP promoters. MEF cells, which were infected with Ad-sh-mSGO2-KD1, Ad-sh-mHSF1-KD2, or Ad-sh-SCR, were treated with heat shock at 42°C for 0, 5, and 30 min. ChIP-qPCR analyses on promoters of HSP70.3 (HSPA1A), HSP40 (DNAJB1), and HSP25 (HSPB1) were performed (left). Extracts of cells were subjected to immunoblotting (right). HSF1 mutants lacking the d-region did not in
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