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• W2001859716 abstract "Article24 September 2009free access PARP-1 transcriptional activity is regulated by sumoylation upon heat shock Nadine Martin Nadine Martin Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: Cell Proliferation Group, MRC Clinical Sciences Centre, London W120NN, UK Search for more papers by this author Klaus Schwamborn Klaus Schwamborn Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: Pepscan Therapeutics BV, Lelystad 8219 PK, The Netherlands Search for more papers by this author Valérie Schreiber Valérie Schreiber IREBS-FRE3211, CNRS, Université de Strasbourg, ESBS, Illkirch, France Search for more papers by this author Andreas Werner Andreas Werner Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: ZMBH, University Heidelberg, Heidelberg 69120, Germany Search for more papers by this author Christelle Guillier Christelle Guillier Plate-forme protéomique, Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, FrancePresent address: UMR INRA/CNRS, Université de Bourgogne, Dijon 21 065, France Search for more papers by this author Xiang-Dong Zhang Xiang-Dong Zhang Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, USA Search for more papers by this author Oliver Bischof Oliver Bischof Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Jacob-S Seeler Corresponding Author Jacob-S Seeler Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Anne Dejean Corresponding Author Anne Dejean Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Nadine Martin Nadine Martin Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: Cell Proliferation Group, MRC Clinical Sciences Centre, London W120NN, UK Search for more papers by this author Klaus Schwamborn Klaus Schwamborn Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: Pepscan Therapeutics BV, Lelystad 8219 PK, The Netherlands Search for more papers by this author Valérie Schreiber Valérie Schreiber IREBS-FRE3211, CNRS, Université de Strasbourg, ESBS, Illkirch, France Search for more papers by this author Andreas Werner Andreas Werner Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, FrancePresent address: ZMBH, University Heidelberg, Heidelberg 69120, Germany Search for more papers by this author Christelle Guillier Christelle Guillier Plate-forme protéomique, Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, FrancePresent address: UMR INRA/CNRS, Université de Bourgogne, Dijon 21 065, France Search for more papers by this author Xiang-Dong Zhang Xiang-Dong Zhang Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, USA Search for more papers by this author Oliver Bischof Oliver Bischof Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Jacob-S Seeler Corresponding Author Jacob-S Seeler Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Anne Dejean Corresponding Author Anne Dejean Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France Search for more papers by this author Author Information Nadine Martin1, Klaus Schwamborn1,‡, Valérie Schreiber2,‡, Andreas Werner1, Christelle Guillier3, Xiang-Dong Zhang4, Oliver Bischof1, Jacob-S Seeler 1 and Anne Dejean 1 1Department of Cell Biology and Infection, Nuclear Organisation and Oncogenesis Unit, INSERM U579, Institut Pasteur, Paris, France 2IREBS-FRE3211, CNRS, Université de Strasbourg, ESBS, Illkirch, France 3Plate-forme protéomique, Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, France 4Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, USA ‡These authors contributed equally to this work *Corresponding authors. BCI-ONO-INSERM U579, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France. Tel.: +33 45 6880 86; Fax: +33 145 6889 43; E-mail: [email protected] or Tel.: +33 145 6888 86; Fax: +33 145 6889 43; E-mail: [email protected] The EMBO Journal (2009)28:3534-3548https://doi.org/10.1038/emboj.2009.279 Present address: Pepscan Therapeutics BV, Lelystad 8219 PK, The Netherlands PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Heat shock and other environmental stresses rapidly induce transcriptional responses subject to regulation by a variety of post-translational modifications. Among these, poly(ADP-ribosyl)ation and sumoylation have received growing attention. Here we show that the SUMO E3 ligase PIASy interacts with the poly(ADP-ribose) polymerase PARP-1, and that PIASy mediates heat shock-induced poly-sumoylation of PARP-1. Furthermore, PIASy, and hence sumoylation, appears indispensable for full activation of the inducible HSP70.1 gene. Chromatin immunoprecipitation experiments show that PIASy, SUMO and the SUMO-conjugating enzyme Ubc9 are rapidly recruited to the HSP70.1 promoter upon heat shock, and that they are subsequently released with kinetics similar to PARP-1. Finally, we provide evidence that the SUMO-targeted ubiquitin ligase RNF4 mediates heat-shock-inducible ubiquitination of PARP-1, regulates the stability of PARP-1, and, like PIASy, is a positive regulator of HSP70.1 gene activity. These results, thus, point to a novel mechanism for regulating PARP-1 transcription function, and suggest crosstalk between sumoylation and RNF4-mediated ubiquitination in regulating gene expression in response to heat shock. Introduction The cellular response to sudden environmental stress is characterized by a rapid activation phase, which is invariably followed by attenuation of the response, despite the persistent presence of the inducing signal. Many transcriptional regulatory mechanisms for this involve post-translational modifications, because their usually transient nature permits both rapid amplification and subsequent extinction of the transduced signals. The heat-shock response represents a well-characterized model system for the study of transcriptional responses to environmental stress. In mammals, a major consequence of heat shock is the activation of a number of heat-shock factors (HSFs) that drive the transcriptional activation of heat-shock protein (HSP) genes that encode protein chaperones involved in protecting cellular functions from the deleterious effects of misfolded, aggregated, or mislocalized proteins (Morimoto, 1998, 2008). The factors that impinge on the regulation of HSP genes are, therefore, a subject of intense scrutiny. Among the proteins now known to play a key role in this regulation is the cellular sensor of DNA damage, poly(ADP-ribose) polymerase 1 (PARP-1, reviewed by Schreiber et al, 2006). PARP-1 is the most abundant and founding member of a super-family of proteins defined by their homology to the catalytic domain of PARP-1 that is responsible for the synthesis of linear or branched polymers of ADP-ribose (PAR) from nicotinamide adenine dinucleotide (NAD+; Schreiber et al, 2006; Hakme et al, 2008). Poly(ADP-ribosyl)ation, besides being strongly induced by DNA-damaging agents, such as reactive oxygen (e.g. H2O2), has been shown to exert major effects on chromatin structure and hence on the regulation of, particularly, transcriptionally active loci (for review, see Kraus, 2008). In Drosophila polytene chromosomes, for example, these PAR-containing loci are readily visible as puffs of de-compacted chromatin, thus providing perhaps the most striking evidence for the association of poly(ADP-ribosyl)ation with chromatin de-condensation (Tulin and Spradling, 2003). The concomitant rapid nucleosome loss, even prior to transcriptional onset, from the Drosophila HSP70 promoter region, has been shown to require PARP activity (Petesch and Lis, 2008). On nucleosomal DNA templates, PARP-1 was shown to occupy a position between nucleosomes, consistent with in vivo results showing that PARP-1 and linker histone H1 occupy distinct and mutually exclusive chromosomal regions (Kim et al, 2004; Krishnakumar et al, 2008). Ouararhni et al (2006) have extended these findings by showing that on the HSP70.1 promoter, DNA-bound PARP-1 is held in place and is enzymatically inactive by interaction with the variant histone macroH2A (mH2A). Perturbation of this interaction results in rapid PARP-1 activation and PARP-1 clearance from the HSP70.1 promoter. The precise mechanism for this release, however, is still unclear. In eukaryotes, modification by the ubiquitin (Ub)-like SUMO proteins has been shown to exert profound effects on the activity of numerous transcription factors and cofactors (Verger et al, 2003; Müller et al, 2004; Gill, 2005). Like Ub, SUMO is covalently conjugated to its targets employing a cascade of E1, E2 (Ubc9), and E3 enzymes, including the PIAS (protein inhibitor of activated stats) proteins (Hay, 2005; Geiss-Friedlander and Melchior, 2007). The modification is reversible through the action of de-sumoylating enzymes called SENPs (Mukhopadhyay and Dasso, 2007). SUMO-2 and -3, but not SUMO-1, can form polymeric chains through a specific lysine, K11, which is part of a consensus modification motif (Tatham et al, 2001). While, unlike ubiquitination, sumoylation does not directly target its substrates to proteosomal degradation, recent genetic and biochemical evidence has uncovered an intriguing crosstalk mechanism with the Ub-proteasome system. This mechanism involves ubiquitination and degradation of poly-SUMO-modified proteins by the RING domain-containing Ub ligase of the Slx5/Slx8 (Saccharomyces cerevisiae; Wang et al, 2006; Burgess et al, 2007; Ii et al, 2007; Sun et al, 2007; Uzunova et al, 2007; Xie et al, 2007; Mullen and Brill, 2008), Slx8/Rfp1/2 (Saccharomyces pombe; Kosoy et al, 2007; Prudden et al, 2007; Sun et al, 2007), and RNF4 (mammals; Häkli et al, 2005; Lallemand-Breitenbach et al, 2008; Tatham et al, 2008) family. These SUMO-targeted Ub ligases contain multiple SUMO-interaction motifs (SIMs), thus providing an efficient binding interface only with SUMO substrates bearing SUMO chains (Tatham et al, 2008). To date, only two proteins, PML and PEA3, have been shown to be subject to poly-SUMO-mediated ubiquitination and degradation by RNF4 (Lallemand-Breitenbach et al, 2008; Tatham et al, 2008; Guo and Sharrocks, 2009). A wide range of environmental stresses has been shown to lead to a massive and rapid increase in global sumoylation, preferentially with SUMO-2/SUMO-3, indicating involvement of a large number of protein targets (Saitoh and Hinchey, 2000; Blomster et al, 2009; Golebiowski et al, 2009). In the present work, we demonstrate that heat shock leads to rapid recruitment of the SUMO machinery on the HSP70.1 promoter and induces PIASy-dependent sumoylation of PARP-1, necessary for full activation of the inducible HSP70.1 gene. Furthermore, we show that PARP-1 is subject to heat-shock-induced, RNF4-mediated ubiquitination, and that, like PIASy, RNF4 controls the amount of modified PARP-1 and is necessary for full activation of HSP70.1 transcription. Altogether, these results functionally link two important post-translational modifications in regulating PARP-1-mediated transcriptional activation in response to stress. Results PARP-1 is a direct binding partner of the SUMO E3 ligase PIASy The SUMO E3 ligase PIASy has been shown to play important roles in numerous cellular processes such as senescence, apoptosis, and transcription (Sachdev et al, 2001; Bischof et al, 2006; Sharrocks, 2006). To gain further insight into PIASy function, we used a biochemical purification approach to identify interaction partners of PIASy (Martin et al, 2008). Among the interacting proteins identified were the signalling protein FIP200 (Martin et al, 2008), the DNA-repair factor Ku70, the heat-shock chaperone HSP70, the arginine methyltransferase PRMT5, and PARP-1 (data not shown). Focusing on PARP-1, we confirmed the in vivo interaction between endogenous PIASy and PARP-1 in a reciprocal experiment in which PIASy was co-immunoprecipitated with PARP-1 from HeLa extracts (Figure 1A). Consistent with these in vivo results, immobilized GST–PIASy specifically bound 35S-methionine-labelled PARP-1, but not an unrelated control protein, in an in vitro GST pull-down assay (Figure 1B, top and middle panels, respectively). A similar experiment carried out with purified baculovirus-produced PARP-1 further confirmed this interaction (Figure 1B, lower panel) and indicated that PIASy and PARP-1 interact directly. Figure 1.PIASy interacts with PARP-1. (A) Endogenous PIASy and PARP-1 interact in vivo. HeLa cell lysates were immunoprecipitated (IP) with mouse anti-PARP-1 or control (IgG) antibodies and probed with anti-PARP-1 and anti-PIASy antibodies. WCL, whole-cell lysate, 2% of amount used in IP. (B) PIASy and PARP-1 interact in vitro. Pull-down experiment with GST, GST–PIASy, and 35S-labelled in vitro translated PARP-1 and luciferase or recombinant PARP-1. Bound proteins revealed by autoradiography or by anti-PARP-1 antibody. Input: 20% of amount used in binding assays. (C) PIASy interacts with both unmodified and poly(ADP-ribosyl)ated PARP-1 in vitro. GST pull down with 35S-labelled in vitro translated PARP-1 (WT, wild type; E988K, catalytically inactive) after incubation of PARP-1 in a poly(ADP-ribosyl)ation reaction containing (+) or not (−) NAD. Bound proteins revealed by autoradiography. Input: 20% of amount used in the binding reaction. PAR–PARP-1, poly(ADP-ribosyl)ated PARP-1. (D) Major protein domains of PIASy and PARP-1. SAP, SAF-A/B, Acinus, and PIAS domain; SP-RING, Siz/PIAS-RING domain; AD, acidic domain; ZnF, zinc fingers; BRCT, BRCA1 C-terminus domain. (E) Integrity of PIASy SP-RING domain is required for PARP-1 interaction. Co-immunoprecipitation (IP) with endogenous PARP-1 and WT or C342F (mut) FLAG–HA–PIASy expressed in HeLa cells; WCL, whole-cell lysate, 5% of amount used in IP. (F) PARP-1 N-terminus and auto-modification domains interact with PIASy. Immobilized GST or GST–PARP-1 domains, expressed in HeLa cells, were incubated with 35S-Met-labelled, in vitro-translated PIASy. Bound proteins were revealed by autoradiography (top panel) and purified GST or GST–PARP-1 proteins were detected with anti-GST antibody (bottom panel). Input: 20% of amount used in binding reactions. Download figure Download PowerPoint To assess the impact of poly(ADP-ribosyl)ation on PIASy–PARP-1 interaction, we carried out a similar in vitro binding assay using auto-poly(ADP-ribosyl)ated PARP-1. Both poly(ADP-ribosyl)ated and non-poly(ADP-ribosyl)ated PARP-1 bound immobilized PIASy without apparent discrimination (Figure 1C). In contrast, PIASy–PARP-1 co-immunoprecipitation in vivo was drastically reduced upon induction of poly(ADP-ribosyl)ation by treatment of cells with hydrogen peroxide, an effect that could be reversed by treatment of the cells with the poly(ADP-ribosyl)ation inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1-(2H)-isoquinolinone (DPQ) (Supplementary Figure S1). Given that poly(ADP-ribosyl)ation does not affect PIASy–PARP-1 binding in vitro, these results suggest that stress-induced poly(ADP-ribosyl)ation can modulate the PIASy–PARP-1 interaction in vivo. Whether hydrogen peroxide-induced poly(ADP-ribosyl)ation of other substrates, or sequestration of poly(ADP-ribosyl)ated PARP-1 into PIASy inaccessible subcellular sites accounts for the reduced interaction, remains to be determined. PIASy and PARP-1 contain several well-defined structural domains (Figure 1D). To determine whether the PIASy SP-RING finger domain is involved in the association, we expressed FLAG–HA-tagged PIASy wild-type (WT) or a derivative mutated in the SP-RING finger motif (Cys342Phe, abolishing E3 ligase activity; Bischof et al, 2006) in HeLa cells for immunoprecipitation experiments. Endogenous PARP-1 was detectable only in anti-HA immunoprecipitates from cells overexpressing WT PIASy (Figure 1E, compare lanes 5 and 6), suggesting that the interaction requires the integrity and/or ligase function of the PIASy RING finger. To next map the regions of PARP-1 responsible for interaction with PIASy, a series of PARP-1 truncation mutants fused to GST were expressed in HeLa cells and purified on glutathione beads. As shown in Figure 1F, both the PARP-1 N-terminus that encompasses the DNA-binding domain, as well as the auto-modification (BRCT) domain, bound 35S-labelled PIASy protein in GST pull down assays, suggesting that these domains, either together or separately, are critical for PIASy interaction. PARP-1 is SUMO-modified and PIASy is poly(ADP-ribosyl)ated PARP-1 has been shown to interact with the E2 SUMO-conjugating enzyme Ubc9 (Masson et al, 1997) and, more recently, to be modified by SUMO (Blomster et al, 2009; Golebiowski et al, 2009; Messner et al, 2009), suggesting that PARP-1 could be a substrate for PIASy SUMO E3 ligase activity. To test this, we first confirmed that PARP-1 is modified by SUMO-1 and SUMO-2 in vitro (Figure 2A), and in vivo, under overexpression conditions (Figure 2B, lanes 2 and 5). Moreover, simultaneous overexpression of Ubc9 stimulated (lane 3), whereas Senp1 abrogated (lane 4), SUMO-1 modification of PARP-1. Western blotting of extracts from HeLa cells overexpressing SUMO-1, or left untransfected, showed that endogenous PARP-1 also is SUMO-modified (Figure 2C, lanes 1 and 2). Similarly, immunoprecipitation of untransfected HeLa cell extracts with anti-SUMO-1 (lane 5) and anti-SUMO-2 (lane 8) antibodies, but not control antibodies (lanes 4 and 7), revealed the presence of endogenously SUMO-modified PARP-1. Figure 2.Cross-modification between PIASy and PARP-1. (A) Sumoylation of PARP-1 in vitro. Sumoylation of recombinant PARP-1 with SUMO-1 or SUMO-2 after 15 min reaction revealed with anti-PARP-1 antibody. (B) Sumoylation of PARP-1 in vivo. HeLa cells were cotransfected with the indicated expression vectors and analysed by western blot with anti-FLAG antibody. Composite figure from a multi-lane, single blot with enhanced contrast. (C) PARP-1 is sumoylated in vivo at the endogenous level. Anti-PARP-1 antibody was used in western blotting to probe whole-cell lysates (WCL) or anti-SUMO-1, anti-SUMO-2, or control (HA, IgG) immunoprecipitates (IP) from untransfected HeLa cells (lanes 1, 3–8), or from cells transfected with SUMO-1 (lane 2). (D) PIASy acts as a SUMO E3 ligase for PARP-1 in vitro. In vitro sumoylation of 35S-labelled in vitro translated (IVT) PARP-1 in the absence (−) or presence (+) of ATP or of PIASy, added as GST fusion or as FLAG eluate from HeLa cells transfected with FLAG–PIASy (Y) or empty vector (C). Reaction products were detected by autoradiography. (E) PIASy acts as a SUMO E3 ligase for PARP-1 in vivo. Lysates from HeLa cells transfected with the indicated expression vectors were probed with anti-FLAG antibody. (F) PARP-1 poly(ADP-ribosyl)ates PIASy in vitro. FLAG eluates from mock-transfected (C) or FLAG–PIASy-transfected (Y) HeLa cells were used as substrates in a poly(ADP-ribosyl)ation reaction catalysed by recombinant PARP-1 and 32P-NAD+. After SDS–PAGE, reaction products were visualized by autoradiography (top panel) and identified by western blotting as indicated (middle and bottom panels). Download figure Download PowerPoint To then test whether PIASy acts as a SUMO E3 ligase for PARP-1, we added bacterially produced GST–PIASy to an in vitro sumoylation reaction. As shown in Figure 2D, GST–PIASy enhanced the sumoylation of PARP-1 by both SUMO-1 (compare lanes 2 and 3) and SUMO-2 (compare lanes 7 and 8). Similarly, use of FLAG–PIASy, expressed in HeLa cells and immunoprecipitated with anti-FLAG antibody, also led to marked enhancement of PARP-1 modification by SUMO-1 (lane 5) and SUMO-2 (lane 10), whereas a mock eluate had no effect (lanes 4 and 9). To confirm these results in vivo, we next coexpressed FLAG–PARP-1 together with PIASy and SUMO. PIASy stimulated the modification by both SUMO isoforms (Figure 2E, lanes 3 and 5). PIAS1, PIASxα, and PIASxβ, but not PIAS3, also showed a stimulating effect on PARP-1 sumoylation in vitro. Moreover, PIASxα also interacts with PARP-1 in vivo (Supplementary Figure 2), suggesting that other PIAS family members may function as SUMO E3 ligases for PARP-1 under these experimental conditions. The finding that PIASy mediates sumoylation of PARP-1 prompted us to investigate whether, reciprocally, PARP-1 could poly(ADP-ribosyl)ate PIASy. For this, we incubated a FLAG–PIASy eluate with 32P-labelled NAD+ and DNAseI-treated DNA in the presence or absence of recombinant PARP-1. As seen in Figure 2F, PARP-1 was efficiently auto-poly(ADP-ribosyl)ated under these conditions (top and middle panels, lanes 2 and 4). Addition of FLAG–PIASy eluate led to the appearance of a second major band corresponding in size to (ADP-ribosyl)ated PIASy (top panel, lane 4). Remarkably, even in the absence of added recombinant PARP-1, weaker signals corresponding to (ADP-ribosyl)ated PIASy (lane 3) and PARP-1 (top and middle panels, lane 3) could be detected, suggesting the presence of endogenous PARP-1 activity in the FLAG–PIASy eluate. Finally, a GST–PIASy fusion protein could also be (ADP-ribosyl)ated by recombinant PARP-1 (data not shown). Taken together, these results indicate that PIASy and PARP-1 cross modify each other, suggesting a possible interplay between these two types of protein modifications. Lysine 486 and 203 are the principal SUMO-acceptor sites of PARP-1 Inspection of the human PARP-1 amino-acid sequence revealed the presence of numerous (>20) putative sumoylation sites, of which five conformed most faithfully to the classical ΨKxE motif (Rodriguez et al, 1999; Figure 3A). One of these (K486) could be confirmed by mass spectroscopy analysis (data not shown). Mutation of these lysine residues to arginine showed two of these, K486 and K203, to be critical for PARP-1 sumoylation, although mutation of either alone, or both (2KR), failed to abolish PARP-1 sumoylation entirely, both in vitro (Figure 3B and Supplementary Figure S3) and in vivo (Figure 3C). Of note, in vitro modification with SUMO-1 (Figure 3B, odd-numbered lanes) revealed the existence of additional sites, whereas modification with SUMO-2 additionally led to the formation of high-molecular-weight (MW) polymeric SUMO-2 chains (lanes 6 and 8). Taken together, these results show PARP-1 to be SUMO-modified on lysine 486 and 203, as well as on other, non-consensus or promiscuous modification sites. Figure 3.Mapping of the SUMO-acceptor sites of PARP-1. (A) Five most probable ΨKxE sumoylation consensus motifs of human PARP-1 protein. (B) In vitro sumoylation of 35S-labelled in vitro translated WT, K203R, K486R, or K203R/K486R (2KR) PARP-1 with SUMO-1 or SUMO-2 revealed by autoradiography after long reaction time (60 min). (C) Sumoylation of FLAG–PARP-1 WT or 2KR mutant in vivo. Lysates from HeLa cells transfected with the indicated expression vectors were probed with anti-FLAG antibody. Download figure Download PowerPoint Heat shock induces PARP-1 sumoylation Environmental stresses such as heat shock, osmotic, or oxidative stress are known to induce the preferential conjugation of SUMO-2/SUMO-3 to numerous target proteins (Saitoh and Hinchey, 2000). In addition, PARP-1 was shown to regulate the expression of the heat-shock-inducible HSP70.1 gene (Ouararhni et al, 2006). These findings prompted us to examine whether heat shock could induce the sumoylation of PARP-1. Consistent with recently published results (Blomster et al, 2009; Golebiowski et al, 2009), coexpression of FLAG–PARP-1 and Ubc9 together with either SUMO-1 or SUMO-2 in HeLa cells exposed to heat shock (43°C, 30 min) resulted in the appearance of slower migrating PARP-1 species in the presence of SUMO-2 but not of SUMO-1 (Figure 4A). In contrast, simultaneous coexpression of SENP6, a de-sumoylating enzyme with specificity for poly-SUMO chains (Mukhopadhyay et al, 2006), led to disappearance of these high-MW PARP-1 species, demonstrating that heat shock promotes the formation of PARP-1–poly-SUMO-2 conjugates (Figure 4B, compare lanes 3 and 4), the abundance of which was significantly reduced when the PARP-1 2KR mutant was expressed instead of WT (Figure 4C). Figure 4.Heat shock induces PIASy-dependent sumoylation of PARP-1. (A) Heat shock induces preferential SUMO-2 modification of PARP-1. Whole-cell lysates of HeLa cells transfected as indicated, untreated or heat shocked (30 min, 43°C), were western blotted with anti-FLAG antibody. (B) Heat shock induces poly-modification of PARP-1 by SUMO-2. Whole-cell lysates of HeLa cells transfected as indicated, untreated or heat shocked (30 min, 43°C), were western blotted with anti-FLAG antibody. Composite figure from a multi-lane, single blot with enhanced contrast. (C) High sumoylation of PARP-1 upon heat shock is impaired by the K203R/K486R (2KR) mutation. Whole-cell lysates of HeLa cells transfected as indicated, untreated or heat shocked (30 min, 43°C), were western blotted with anti-FLAG antibody. (D) Modified PARP-1 partitions to the detergent-insoluble fraction. HeLa cells transfected as indicated, untreated, or heat shocked (30 min, 43°C) were either lysed directly in SDS sample buffer (TCE, total cell extract, left panel), or extracted in NP-40-containing Chris buffer, separated into soluble (Sol.) and insoluble (Insol.) fractions by centrifugation (right panels), and western blotted with anti-FLAG antibody. (E) Heat shock induces accumulation of modified forms of endogenous PARP-1 in HeLa cells. Whole-cell lysates of HeLa cells, untreated or heat shocked (30 min, 43°C), were western blotted with anti-PARP-1 antibody. (F) Heat shock induces sumoylation of endogenous PARP-1 in HeLa cells. FLAG (control) or PARP-1 immunoprecipitates from untransfected HeLa cells untreated or heat shocked (30 min, 43°C) were analysed by western blot using anti-SUMO-2 antibody (right panel). The corresponding whole-cell lysates were analysed using anti-PARP-1 antibody (left panel). (G) PIASy enhances heat-shock-induced PARP-1 sumoylation. HeLa cells were transfected as indicated and left untreated or heat shocked (30 min, 43°C). Whole-cell lysates were probed with the indicated antibodies. (H) PIASy is required for the heat-shock-induced increase in PARP-1 sumoylation. HeLa cells were transfected with scrambled control (scr) or PIASy siRNA and re-transfected 24 h later with FLAG–PARP-1, SUMO-2, and Ubc9. After 24 h, cells were either left untreated or heat shocked (30 min, 43°C). Whole-cell lysates were probed with the indicated antibodies. Download figure Download PowerPoint Fractionation of cell extracts from PARP-1-, Ubc9-, and SUMO-2-overexpressing cells further revealed enhanced association of modified PARP-1 with the detergent (Nonidet P-40 (NP-40))-insoluble fraction under heat shock (Figure 4D), suggesting that the induced sumoylation of PARP-1 is preferentially associated with the chromatin and/or nuclear matrix compartment. Non-transfected HeLa cells similarly displayed accumulation of modified endogenous PARP-1 species upon heat shock (Figure 4E). Immunoprecipitation with anti-PARP-1 antibody (Figure 4F, lanes 4 and 6), or anti-FLAG control antibody (lanes 3 and 5), from extracts of unstressed or heat shocked HeLa cells confirmed that these endogenous higher-MW PARP-1 species correspond to polymeric or multiply modified PARP-1–SUMO-2 conjugates. Consistent with previous in vitro results, overexpression of PIASy (Figure 4G), PIASxα, or PIASxβ (Supplementary Figure S2B, lanes 10 and 11) stimulated heat-shock-induced PARP-1 sumoylation under cotransfection conditions. Conversely, siRNA-mediated knockdown of PIASy expression in HeLa cells almost completely abolished the heat-shock-induced sumoylation of PARP-1 (Figure 4H, compare lanes 5 and 6), whereas cells transfected with a scrambled control siRNA behaved like mock-transfected cells (compare lanes 4 and 5), suggesting that PIASy occupies a privileged position as a SUMO E3 ligase for PARP-1 under heat shock. Taken together, these results show that heat shock strongly upregulates PARP-1 sumoylation, in both quantity as well as quality (SUMO-2 polymers), and further, that PIASy appears to play a critical role in this process in vivo. Role of PARP-1 sumoylation in HSP70.1-promoter activation Given the role of PARP-1 in the transcriptional regulati" @default.
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• W2001859716 title "PARP-1 transcriptional activity is regulated by sumoylation upon heat shock" @default.
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