- Email: [email protected]
ZNF746 regulates its transcriptional activity
Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
SUMOylation of the KRAB zinc-finger transcription factor PARIS/ ZNF746 regulates its transcriptional activity Tamotsu Nishida*, Yoshiji Yamada Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2016 Accepted 11 April 2016 Available online 14 April 2016
Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. © 2016 Elsevier Inc. All rights reserved.
Keywords: SUMO PARIS PIASy PGC-1a HDAC
1. Introduction Krüppel-associated box (KRAB)-containing zinc-finger proteins (KRAB-ZFPs) comprise the largest family of transcription factors in mammals [1,2]. KRAB-ZFPs play a role in the regulation of diverse cellular functions, including cell differentiation, proliferation, and apoptosis [1,3]. The zinc-finger motif regulates sequence specific DNA binding to regulatory elements present in the promoter regions of target genes, while the KRAB domain mediates transcriptional repressor activity [4,5], which is dependent on the interaction with a co-repressor KRAB-associated protein 1 (KAP-1), also known as TIF1b or KRIP-1 [6e8]. KAP-1 recruits chromatin modifying protein complexes, such as the NuRD histone deacetylase complex and the histone H3 lysine 9-specific methyltransferase SETDB1, which promotes binding of heterochromatin protein 1
Abbreviations: PARIS, Parkin-interacting substrate; SUMO, Small ubiquitinrelated modifier; KRAB, Krüppel-associated box; ZFPs, zinc-finger proteins; PGC1a, peroxisome proliferator-activated receptor g coactivator-1a; DBD, DNA-binding domain; PIAS, protein inhibitor of activated STAT; HDAC, histone deacetylase; TSA, trichostatin A; EGFP, enhanced green fluorescent protein. * Corresponding author. E-mail address: [email protected] (T. Nishida). http://dx.doi.org/10.1016/j.bbrc.2016.04.051 0006-291X/© 2016 Elsevier Inc. All rights reserved.
(HP1) to specific binding sites of KRAB-ZNFs, leading to the formation of heterochromatin and transcriptional repression [9e11]. KRAB-ZFPs, which act as potent transcriptional repressors of their target genes, are well characterized; however, their gene targets in vivo or physiological functions and post-translational modification-related regulatory mechanisms remain poorly understood. Reversible post-translational modifications such as phosphorylation play a pivotal role in the regulation of protein function in eukaryotes. Small ubiquitin-related modifier (SUMO) modification (SUMOylation) is a form of post-translational modification that regulates the function of substrate proteins involved in normal cellular processes [12,13]. Aberrations in the SUMOylation pathway are implicated in the pathogenesis of human diseases such as cancer and neurodegeneration [14]. SUMOylation occurs in a manner analogous to the ubiquitination reaction and requires a heterodimeric E1-activating enzyme, consisting of an Aos1/Uba2 heterodimer, an E2-conjugating enzyme, Ubc9, and an E3 ligase that mediates the conjugation of SUMO from the E2 to its target protein; well characterized SUMO E3 ligases include protein inhibitor of activated STAT (PIAS) family members RanBP2 and polycomb group protein Pc2/ CBX4 [13]. SUMOylation is a highly dynamic process that can be reversed by SUMO-specific proteases [15].
1262
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
PARIS (also known as ZNF746), a member of the family of KRABZFPs transcriptional repressors, is a substrate of the ubiquitin E3 ligase parkin, a gene product associated with autosomal recessive Parkinson's disease (PD) [16]. Inactivation of parkin leads to accumulation of PARIS in PD brain cells. PARIS represses the expression of the transcriptional co-activator PGC-1a, which controls the expression of many genes involved in energy metabolism and mitochondrial biogenesis [17]. The parkin-PARIS-PGC-1a pathway is thought to contribute to PD pathogenesis [16,18]. However, the molecular mechanisms controlling the transcriptional activity of PARIS remain largely unclear. In the present study, we showed that PARIS can be SUMOylated and its SUMOylation influences its transcriptional activity. We also showed that PIASy interacts with PARIS and promotes its SUMOylation, whereas it represses the PGC-1a promoter through a PARISindependent mechanism. The present data may improve our understanding of the role of SUMOylation in neurodegeneration in PD. 2. Materials and methods 2.1. Plasmid constructs and siRNA For transient expression of recombinant PARIS proteins in human cell lines, the human PARIS cDNA was amplified by RT-PCR using total mRNA from SH-SY5Y cells, and cloned into pFLAGCMV-2 (SigmaeAldrich, St. Louis, MO), pcDNA3.1/Myc (Invitrogen, Carlsbad, CA) or pM (Clontech, Mountain View, CA), which expresses the GAL4-DNA binding domain (GAL4-DBD) fusion protein. The substitution or deletion mutants of PARIS were created by a PCR-based mutation method using specific mutation primers. SUMO-1, SENP1, and PIAS expression plasmids were described previously [19]. For the reporter assay, the human PGC-1a gene promoter fragment (992 to þ90) was amplified by PCR with the primers 50 -ggggtaccGCTAATAGTGTGTTGGTATTTT-30 and 50 0 TCCTGAATGACGCCAGTCAAGC-3 . This fragment was digested with KpnI/HindIII and then cloned into the KpnI/HindIII site of the pGL3basic vector (designated pGL3-PGC-1a). pL8G5-Luc, pL8-Luc, and pLexA-VP16 were kindly provided by Dr. S. Khochbin [20]. Validated Stealth™ RNAi duplex targeting human PIAS4 (PIASy) and Stealth™ RNAi negative control duplex were purchased from Invitrogen. 2.2. Cell culture and transfection All human cell lines used in this study were maintained in Dulbecco's Modified Eagles' Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. 2.3. Co-immunoprecipitation and immunoblotting Preparation of cell lysates, co-immunoprecipitation, and immunoblotting were performed as described previously [19]. 2.4. Reporter assays Luciferase reporter assays were performed as described previously [19]. Briefly, cells were co-transfected with the reporter construct pRL-CMV, expressing Renilla luciferase, as an internal control, and protein expression constructs, as indicated. At 48 h after transfection, luciferase activities were determined by using a dual luciferase reporter assay system (Promega, Madison, WI) and a GloMax 20/20 luminometer (Promega) according to the manufacturer's instructions. Luciferase activity was normalized to Renilla
luciferase activity from the pRL-CMV vector. The results were presented as the means and standard deviation for relative luciferase activity from at least three independent experiments. 3. Results 3.1. PARIS can be SUMOylated In vivo SUMOylation assays were performed to determine whether PARIS can be SUMOylated in cells. In cells co-transfected with FLAG-PARIS and EGFP-SUMO-1, the FLAG antibody recognized a major 100-kD band (the expected size of the unmodified form of FLAG-PARIS) and two additional slower-migrating 220- and 270-kD bands (Fig. 1A, lane 2). Co-expression of the SUMO-specific protease SENP1 abolished the slower-migrating bands (Fig. 1A, lane 3 and 4), but not the 100-kD band, suggesting that the two slowermigrating bands represent SUMOylated forms of PARIS. PARIS was SUMOylated by SUMO-2/3, but less efficiently than by SUMO-1 in cells (Supplementary Fig. S1). SUMOylation of PARIS was confirmed in vitro (Supplementary Fig. S2). Although SUMO-1 conjugation was more efficient in vivo, the conjugation of SUMO-1 and SUMO-2/3 to PARIS was equally efficient in vitro. 3.2. PARIS is mainly SUMOylated at K189 and K286 SUMOylation typically occurs within the consensus sequence
JKXE, where J represents a large hydrophobic amino acid, K is the lysine conjugated to SUMO, X is any amino acid, and E is a glutamic acid [21]. Analysis of the amino acid sequence of human PARIS revealed that K189 and K286 are located within perfectly matching consensus sequences (Fig. 1B and C). To verify that these lysines were SUMOylation sites, we generated mutants replacing lysine with arginine (KR mutants) or glutamic acid within consensus sequences with alanine (EA mutants), and performed in vivo SUMOylation assays using these mutants (Fig. 1D). Single point mutations (K189R, K286R, E191A or E288A) abolished only the slowest-migrating 270-kD SUMOylated form (lanes 3, 4, 6 and 7). Both double mutants, K189R/K286R (2KR) and E191A/E288A (2EA), completely lost the SUMOylation of PARIS (lanes 5 and 8). The loss of SUMOylation bands in the 2KR mutant was confirmed in vitro (Supplementary Fig. S3). These results indicated that K189 and K286 are the primary SUMOylation sites in the human PARIS protein. 3.3. SUMOylation regulates the transcriptional activity of PARIS PARIS represses the expression of the transcriptional coactivator PGC-1a by binding to insulin response sequences (IRS) in the PGC-1a promoter region in SH-SY5Y cells [16]. We therefore investigated the effect of SUMOylation of PARIS on the PARISmediated repression of PGC-1a promoter activity in cells. For this purpose, reporter assays were performed in four human cell lines co-transfected with pGL3-PGC-1a and wild-type or the 2KR mutant of PARIS (Fig. 2A). In SH-SY5Y cells and H1299 cells, wild-type PARIS repressed the reporter activity as previously reported, and this effect was partially restored by transfection with the 2KR mutant. The 2EA mutant showed almost the same repression activity as the 2KR mutant (data not shown). In HepG2 cells, wild-type PARIS had little effect on reporter activity, whereas cells transfected with the 2KR mutant exhibited significantly higher transcriptional activity than those expressing wild-type PARIS. Conversely, in HEK293 cells, wild-type PARIS activated, whereas 2KR mutant repressed the reporter activity. These results indicated that the effects of PARIS and its SUMOylation on PGC-1a promoter activity are cell type dependent. Next, we investigated the role of the KRAB domain of PARIS in
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
1263
Fig. 1. PARIS is SUMOylated at K189 and K286 in vivo. (A) PARIS can be modified by SUMO-1 in vivo. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS, EGFP-SUMO-1, and Myc-SENP1 [wild-type (WT) or the catalytically inactive mutant C599A (CA)] as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (B) Schematic representation of the human PARIS protein. Its known domains and two lysines (K189 and K286) in the consensus motif for SUMOylation are indicated. (C) Alignment of the sequences surrounding two putative SUMOylation sites (K189 and K286) in the human PARIS protein. (D) K189 and K286 in PARIS are major SUMOylation sites in vivo. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS (WT or the indicated mutants) and EGFP-SUMO-1 as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-FLAG antibody.
Fig. 2. SUMOylation affects the transcriptional activity of PARIS. (A) Effects of SUMOylation of PARIS on PGC-1a promoter activity in different human cell lines. Cells were cotransfected with pGL3-PGC-1a or its control, pGL3-Basic (400 ng), together with expression constructs for FLAG-PARIS (100 ng, WT or the indicated mutants) as indicated. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with empty vector (taken as 1). A schematic representation of the human PGC-1a gene promoterreporter construct is shown at the top. (B) Effects of SUMOylation of PARIS on the L8G5 reporter activity in different human cell lines. Cells were co-transfected with pL8G5-Luc or its control, pL8-Luc (400 ng), together with expression constructs for GAL4-DBD-PARIS (100 ng, WT or the indicated mutants) in the presence or absence of pLexA-VP16 (100 ng) as indicated. The data are expressed relative to the luciferase activity of the pL8G5-Luc co-transfected with control vector pM expressing the GAL4-DBD alone in the presence of pLexAVP16 (taken as 1). A schematic representation of the L8G5-Luc chimeric promoter-reporter construct is shown at the top.
the regulation of PGC-1a promoter activity by generating a KRAB domain deletion mutant (DKRAB). Our results showed that the DKRAB mutant was SUMOylated in cells (data not shown) and
markedly increased reporter activity in all cell lines tested (Fig. 2A). The addition of the 2KR mutation to the DKRAB mutant (DKRAB/ 2KR) reduced the activity to levels similar to those of the 2KR
1264
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
Fig. 3. PIASy enhances the SUMOylation of PARIS in vivo. (A) PIASy enhances the SUMOylation of PARIS by all SUMO isoforms. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS, EGFP-SUMO (SUMO-1, SUMO-2, or SUMO-3) and Myc-PIASy as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (B) PIASy interacts with PARIS in vivo. The cell lysates of COS-7 cells co-expressing FLAG-PARIS (WT or 2KR) and Myc-PIASy WT (top panels), or FLAG-PARIS WT and Myc-PIASy [WT or the C342A mutant (CA)] (bottom panels) as indicated were immunoprecipitated (IP) with anti-FLAG antibody. The precipitates and an aliquot of the cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.
mutant. Similar results were observed with a DKRAB mutant containing a 2EA mutation (data not shown). Although the molecular mechanism remains unclear, these data indicated that SUMOylation of PARIS may be required for its transcriptional activation potential. To examine the function of PARIS in transcription, GAL4-DBDPARIS fusions were constructed and co-transfected with a LexAGal4-driven promoter reporter plasmid (L8G5-Luc) in which the reporter was activated by a fusion protein of the LexA DNA-binding domain and the VP16 activation domain (LexA-VP16) [20]. In the control experiments, L8G5-Luc was substituted by an L8-Luc reporter lacking Gal4-binding sites. These reporters were activated only in the presence of the LexA-VP16 fusion protein (Fig. 2B). In all cell lines tested, wild-type PARIS significantly repressed the LexAVP16-induced reporter activity in a GAL4-binding site-dependent manner, suggesting that PARIS functions as a transcriptional repressor. On the other hand, the 2KR mutant significantly increased the LexA-VP16-induced reporter activity compared with that in controls. In contrast to the reporter assays using the PGC-1a promoter construct, the DKRAB mutation had no effect on the potential activity of PARIS. These results suggested that PARIS has the potential to both activate and repress transcription, and that SUMOylation of PARIS functions in transcriptional repression. SUMOylation of PARIS may switch its role from that of an activator to that of a repressor.
3.4. The SUMO E3 ligase PIASy promotes the SUMOylation of PARIS Next, we searched for the SUMO E3 ligase that specifically SUMOylates PARIS. Several SUMO E3 ligases have been identified to date, and PIAS family members are well known SUMO E3 ligases that target transcription factors [22e24]. Therefore, we examined
the effect of PIAS family proteins on the SUMOylation of PARIS using in vivo SUMOylation assays. Among five PIAS family members analyzed, only PIASy significantly enhanced the SUMOylation of PARIS by all SUMO isoforms and particularly increased the efficiency of SUMO-2/3 conjugation (Supplementary Fig. S4 and Fig. 3A, lanes 3, 5, and 7). To determine whether PIASy interacts with PARIS, COS-7 cells were co-transfected with expression constructs for FLAG-PARIS and Myc-PIASy and analyzed by coimmunoprecipitation. The results showed that PIASy specifically interacted with PARIS (Fig. 3B, lane 3 in each panel), and the 2KR mutation of PARIS or the RING domain mutation, which abolishes the SUMO E3 ligase activity of PIASy, did not affect their interaction (lane 4 in each panel). 3.5. PIASy represses PGC-1a promoter activity PIAS family proteins have both positive and negative effects the activity and expression of many transcription factors in either a SUMOylation-dependent or -independent manner [22e24]. We therefore investigated the effects of PIASy on PGC-1a promoter transcription. SH-SY5Y cells were co-transfected with a PGC-1a promoter reporter together with an expression construct for either wild-type or a SUMO E3 ligase activity-deficient mutant (CA) of PIASy and/or SUMO-1. As shown in Fig. 4A, the expression of wildtype PIASy significantly reduced the reporter activity (lane 4), whereas SUMO-1 alone slightly decreased the activity (lane 3). Coexpression of SUMO-1 with PIASy clearly enhanced the repression of the reporter activity by wild-type PIASy (lane 5). The CA mutant repressed reporter activity less effectively than the wild type PIASy (lane 6), whereas it abolished the enhancing effect of SUMO-1 coexpression on transcriptional repression (lane 7). Conversely, siRNA-mediated knockdown of PIASy in SH-SY5Y cells significantly
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
1265
Fig. 4. Effect of PIASy on PARIS-mediated repression of PGC-1a promoter activity. (A) PIASy represses PGC-1a promoter activity. SH-SY5Y cells were co-transfected with pGL3PGC-1a or its control, pGL3-basic (400 ng), together with expression constructs for FLAG-PIASy [100 ng, WT or C342A (CA)] and EGFP-SUMO-1 (100 ng) as indicated. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with empty vectors (taken as 1). (B) Effects of PIASy knockdown on PGC-1a promoter activity. SH-SY5Y cells were co-transfected with pGL3-PGC-1a (400 ng), together with negative control siRNA (40 pmol), PIASy-siRNA (40 pmol) or vehicle (non-siRNA control) as indicated. The data are expressed relative to the luciferase activity in the presence of negative control siRNA duplexes (taken as 1). Immunoblotting of cell lysates from the luciferase assay was performed to confirm the knockdown of PIASy proteins. b-actin was used as the loading control (bottom panels). (C) PIASy attenuates the PARIS-mediated repression of PGC-1a promoter activity in a SUMOylation status-dependent manner. SH-SY5Y cells were co-transfected with pGL3-PGC-1a or its control, pGL3-basic (400 ng), together with expression constructs for FLAG-PIASy WT (100 ng), FLAG-PARIS (100 ng, either WT or 2KR) and EGFP-SUMO-1 (100 ng) as indicated. Cells were incubated for 48 h and analyzed for luciferase activity. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with pRL-CMV and empty vectors (taken as 1). (D) PIASy-mediated repression of PGC-1a promoter activity is independent of HDAC activity. SH-SY5Y cells were co-transfected with pGL3-PGC-1a (400 ng), in the absence or presence of expression constructs for FLAG-PARIS (100 ng) and/or FLAG-PIASy (100 ng) as indicated. At 24 h after the transfection, cells were treated with increasing doses of trichostatin A (TSA) for an additional 24 h and then analyzed for luciferase activity. The data are expressed relative to the luciferase activity in the absence of TSA (taken as 1).
increased the reporter activity (Fig. 4B, lane 3). These results suggested that PIASy is involved in the repression of the PGC-1a promoter in either a SUMOylation-dependent or -independent manner. 3.6. PIASy influences PARIS-mediated repression of PGC-1a promoter activity Next, we examined the effects of expression of PIASy on the PARIS-mediated repression of PGC-1a promoter activity in SH-SY5Y cells (Fig. 4C). Co-transfection with the PGC-1a promoter reporter together with an expression construct for either PIASy or wild-type PARIS significantly decreased reporter activity (lanes 4 and 6). However, co-expression of PIASy with wild-type PARIS, but not with the 2KR mutant, attenuated its repressive activity (lanes 8 and 12). By contrast, co-expression of PIASy with the 2KR mutant synergistically repressed the reporter activity, although the 2KR mutant alone did not repress the reporter activity. Co-expression
SUMO-1 with PIASy completely repressed the reporter activity independently of PARIS (lanes 5, 9, and 13). These results indicated that PIASy affects the PARIS-mediated repression of PGC-1a promoter activity in a manner dependent on the SUMOylation status of PARIS, and that PIASy-mediated repression predominates over PARIS activity in SUMO-1 overexpressing cells. 3.7. PIASy-mediated repression of PGC-1a promoter activity is independent of HDAC activity PIASy represses the activity of several transcription factors and co-factors by recruiting HDACs [25,26]. Therefore, we examined whether HDAC activity is required for PIASy-mediated repression of PGC-1a promoter activity. SH-SY5Y cells were co-transfected with a PGC-1a promoter reporter together with or without PIASy and/or PARIS, and treated with increasing amounts of trichostatin A (TSA), an HDAC inhibitor, followed by determination of reporter activities. As shown in Fig. 4D, treatment with increasing doses of TSA
1266
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
increased PGC-1a promoter activity in control and PARIS alone transfected cells (top and second panels). However, TSA had almost no effect on PIASy-mediated repression of PGC-1a promoter activity in the absence and presence of PARIS (third and bottom panels). These results suggested that PARIS-mediated repression, but not PIASy-mediated repression, depends on HDAC activity, and that PIASy does not recruit a repressor complex with HDAC activity to the PGC-1a promoter. 4. Discussion In the present study, we demonstrated that PARIS can be modified by SUMO, and that its SUMOylation plays an important role in the regulation of its transcriptional activity. A SUMOylation deficient mutant restored the repression of PGC-1a promoter activity in SH-SY5Y cells, suggesting that the SUMOylation of PARIS plays a role in its repressive activity. However, our results suggested that the PARIS-mediated regulation of PGC-1a promoter activity is more complex. Reporter analyses showed that the effect of PARIS on the PGC-1a promoter varied significantly in a cell type dependent manner. The fact that the PGC-1a promoter fragment (992 to þ90) contains several consensus transcription factor binding sites, including GC-box, CRE, IRS, SRE, and E-Box [27], indicates that PARIS may interact with other transcription factors that bind to these sites on the promoter. Moreover, co-factors binding to PARIS and/or other transcription factors may affect the ability of PARIS to regulate the PGC-1a promoter activity. The effect of PARIS on the PGC-1a promoter could be affected by basal levels of expression of other transcription factors and/or cofactors in different cell types. Similar to other KRAB-ZFPs, the KRAB domain of PARIS played an important role in its transcriptional repression activity. The DKRAB mutant enhanced PGC-1a promoter activity, whereas it had no effect on LexA-VP16-induced promoter activity, suggesting that the KRAB-mediated interaction of PARIS with co-factor(s) plays a role in PGC-1a promoter repression. KAP-1 is an essential co-factor of KRAB-ZFPs [28]. The interaction of KAP-1 with KRAB-ZNFs promotes the recruitment of a repression complex [9e11]. We investigated the interaction of KAP-1 with PARIS by co-immunoprecipitation assays and its effect on PARIS-mediated transcriptional repression of the PGC-1a promoter was analyzed using reporter assays. An interaction between KAP-1 and PARIS was not detected in the present study. Moreover, KAP-1 overexpression or knockdown in SH-SY5Y cells did not significantly affect the PARIS-mediated transcriptional repression (data not shown). However, we cannot completely rule out the possibility that KAP-1 is involved in the repressive activity of PARIS. Our results showed that PIASy acts as a SUMO E3 ligase for PARIS and is involved in the repression of the PGC-1a promoter in SH-SY5Y cells. Expression of PIASy alone repressed PGC-1a promoter activity in the absence of PARIS (Fig. 4A). Co-expression of PIASy and PARIS significantly attenuated their mutually repressive effect, although this attenuation was compromised by the SUMOylation-deficient mutation of PARIS or co-expression of SUMO-1 (Fig. 4C). The PIASy repression of PGC-1a promoter activity occurred through an HDAC-independent mechanism, whereas the repressive activity of PARIS markedly depended on HDAC activity (Fig. 4D). Taken together, these findings suggest that PIASy-mediated SUMOylation and interaction do not contribute to the repressive activity of PARIS. However, the relatively modest and transient SUMOylation of PARIS may be necessary to attenuate the repression of the PGC-1a promoter by co-expression of PIASy. Further studies are needed to improve our understanding of the molecular mechanisms underlying the PARIS-mediated regulation of PGC-1a promoter activity.
Conflicts of interest The authors declare no conflicts of interest. Acknowledgements We thank Dr. Saadi Khochbin (University Grenoble-Alpes, France) for providing reporter plasmids, and Dr. Shinji Oikawa (Mie University, Japan) for providing SH-SY5Y cells. This work was supported in part by Grants-in-Aid for the Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant Numbers 24500406 and 15K06738 to T.N.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.04.051. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.051. References [1] R. Urrutia, KRAB-containing zinc-finger repressor proteins, Genome Biol. 4 (2003) 231e238. [2] S. Huntley, D.M. Baggott, A.T. Hamilton, M. Tran-Gyamfi, S. Yang, J. Kim, L. Gordon, E. Branscomb, L. Stubbs, A comprehensive catalog of human KRABassociated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors, Genome Res. 16 (2006) 669e677. [3] A. Lupo, E. Cesaro, G. Montano, D. Zurlo, P. Izzo, P. Costanzo, KRAB-Zinc finger proteins: a repressor family displaying multiple biological functions, Curr. Genomics 14 (2013) 268e278. [4] J.F. Margolin, J.R. Friedman, W.K. Meyer, H. Vissing, H.J. Thiesen, F.J. Rauscher 3rd, Krüppel-associated boxes are potent transcriptional repression domains, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4509e4513. [5] R. Witzgall, E. O'Leary, A. Leaf, D. Onaldi, J.V. Bonventre, The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4514e4518. [6] J.R. Friedman, W.J. Fredericks, D.E. Jensen, D.W. Speicher, X.P. Huang, E.G. Neilson, F.J. Rauscher 3rd, KAP-1, a novel corepressor for the highly conserved KRAB repression domain, Genes Dev. 10 (1996) 2067e2078. [7] P. Moosmann, O. Georgiev, B. Le Douarin, J.P. Bourquin, W. Schaffner, Transcriptional repression by RING finger protein TIF1b that interacts with the KRAB repressor domain of KOX1, Nucleic Acids Res. 24 (1996) 4859e4867. [8] S.S. Kim, Y.M. Chen, E. O'Leary, R. Witzgall, M. Vidal, J.V. Bonventre, A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15299e15304. [9] D.C. Schultz, J.R. Friedman, F.J. Rauscher III, Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD, Genes Dev. 15 (2001) 428e443. http://www.ncbi.nlm.nih. gov/pubmed/11230151. comments. [10] D.C. Schultz, K. Ayyanathan, D. Negorev, G.G. Maul, F.J. Rauscher 3rd, SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zincfinger proteins, Genes Dev. 16 (2002) 919e932. http://www.ncbi.nlm.nih. gov/pubmed/11959841. comments. [11] S.P. Sripathy, J. Stevens, D.C. Schultz, The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression, Mol. Cell. Biol. 26 (2006) 8623e8638. [12] F. Melchior, SUMO-nonclassical ubiquitin, Annu. Rev. Cell Dev. Biol. 16 (2000) 591e626. [13] R.T. Hay, SUMO: a history of modification, Mol. Cell 18 (2005) 1e12. [14] K.D. Sarge, O.K. Park-Sarge, Sumoylation and human disease pathogenesis, Trends. Biochem. Sci. 34 (2009) 200e205. [15] D. Mukhopadhyay, M. Dasso, Modification in reverse: the SUMO proteases, Trends. Biochem. Sci. 32 (2007) 286e295. [16] J.H. Shin, H.S. Ko, H. Kang, Y. Lee, Y.I. Lee, O. Pletinkova, J.C. Troconso, V.L. Dawson, T.M. Dawson, PARIS (ZNF746) repression of PGC-1a contributes to neurodegeneration in Parkinson's disease, Cell 144 (2011) 689e702. [17] J.A. Villena, New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond, FEBS J. 282 (2015) 647e672.
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267 [18] D.A. Stevens, Y. Lee, H.C. Kang, B.D. Lee, Y.I. Lee, A. Bower, H. Jiang, S.U. Kang, S.A. Andrabi, V.L. Dawson, J.H. Shin, T.M. Dawson, Parkin loss leads to PARISdependent declines in mitochondrial mass and respiration, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 11696e11701. [19] T. Nishida, M. Terashima, K. Fukami, PIASy-mediated repression of the Ets-1 is independent of its sumoylation, Biochem. Biophys. Res. Commun. 345 (2006) 1536e1546. [20] C. Lemercier, A. Verdel, B. Galloo, S. Curtet, M.P. Brocard, S. Khochbin, mHDA1/ HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity, J. Biol. Chem. 275 (2000) 15594e15599. [21] M.S. Rodriguez, C. Dargemont, R.T. Hay, SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting, J. Biol. Chem. 276 (2001) 12654e12659. [22] D. Schmidt, S. Müller, PIAS/SUMO: new partners in transcriptional regulation, Cell. Mol. Life Sci. 60 (2003) 2561e2574. [23] A.D. Sharrocks, PIAS proteins and transcriptional regulation-more than just SUMO E3 ligases? Genes Dev. 20 (2006) 754e758.
1267
€a €skel€ [24] M.M. Rytinki, S. Kaikkonen, P. Pehkonen, T. Ja ainen, J.J. Palvimo, PIAS proteins: pleiotropic interactors associated with SUMO, Cell. Mol. Life Sci. 66 (2009) 3029e3041. [25] J. Long, I. Matsuura, D. He, G. Wang, K. Shuai, F. Liu, Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 9791e9796. [26] M. Gross, R. Yang, I. Top, C. Gasper, K. Shuai, PIASy-mediated repression of the androgen receptor is independent of sumoylation, Oncogene 23 (2004) 3059e3066. [27] I. Irrcher, V. Ljubicic, A.F. Kirwan, D.A. Hood, AMP-activated protein kinaseregulated activation of the PGC-1a promoter in skeletal muscle cells, PloS One 3 (2008) e3614. [28] M. Abrink, J.A. Ortiz, C. Mark, C. Sanchez, C. Looman, L. Hellman, P. Chambon, R. Losson, Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 b, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1422e1426.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
SUMOylation of the KRAB zinc-finger transcription factor PARIS/ ZNF746 regulates its transcriptional activity Tamotsu Nishida*, Yoshiji Yamada Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2016 Accepted 11 April 2016 Available online 14 April 2016
Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. © 2016 Elsevier Inc. All rights reserved.
Keywords: SUMO PARIS PIASy PGC-1a HDAC
1. Introduction Krüppel-associated box (KRAB)-containing zinc-finger proteins (KRAB-ZFPs) comprise the largest family of transcription factors in mammals [1,2]. KRAB-ZFPs play a role in the regulation of diverse cellular functions, including cell differentiation, proliferation, and apoptosis [1,3]. The zinc-finger motif regulates sequence specific DNA binding to regulatory elements present in the promoter regions of target genes, while the KRAB domain mediates transcriptional repressor activity [4,5], which is dependent on the interaction with a co-repressor KRAB-associated protein 1 (KAP-1), also known as TIF1b or KRIP-1 [6e8]. KAP-1 recruits chromatin modifying protein complexes, such as the NuRD histone deacetylase complex and the histone H3 lysine 9-specific methyltransferase SETDB1, which promotes binding of heterochromatin protein 1
Abbreviations: PARIS, Parkin-interacting substrate; SUMO, Small ubiquitinrelated modifier; KRAB, Krüppel-associated box; ZFPs, zinc-finger proteins; PGC1a, peroxisome proliferator-activated receptor g coactivator-1a; DBD, DNA-binding domain; PIAS, protein inhibitor of activated STAT; HDAC, histone deacetylase; TSA, trichostatin A; EGFP, enhanced green fluorescent protein. * Corresponding author. E-mail address: [email protected] (T. Nishida). http://dx.doi.org/10.1016/j.bbrc.2016.04.051 0006-291X/© 2016 Elsevier Inc. All rights reserved.
(HP1) to specific binding sites of KRAB-ZNFs, leading to the formation of heterochromatin and transcriptional repression [9e11]. KRAB-ZFPs, which act as potent transcriptional repressors of their target genes, are well characterized; however, their gene targets in vivo or physiological functions and post-translational modification-related regulatory mechanisms remain poorly understood. Reversible post-translational modifications such as phosphorylation play a pivotal role in the regulation of protein function in eukaryotes. Small ubiquitin-related modifier (SUMO) modification (SUMOylation) is a form of post-translational modification that regulates the function of substrate proteins involved in normal cellular processes [12,13]. Aberrations in the SUMOylation pathway are implicated in the pathogenesis of human diseases such as cancer and neurodegeneration [14]. SUMOylation occurs in a manner analogous to the ubiquitination reaction and requires a heterodimeric E1-activating enzyme, consisting of an Aos1/Uba2 heterodimer, an E2-conjugating enzyme, Ubc9, and an E3 ligase that mediates the conjugation of SUMO from the E2 to its target protein; well characterized SUMO E3 ligases include protein inhibitor of activated STAT (PIAS) family members RanBP2 and polycomb group protein Pc2/ CBX4 [13]. SUMOylation is a highly dynamic process that can be reversed by SUMO-specific proteases [15].
1262
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
PARIS (also known as ZNF746), a member of the family of KRABZFPs transcriptional repressors, is a substrate of the ubiquitin E3 ligase parkin, a gene product associated with autosomal recessive Parkinson's disease (PD) [16]. Inactivation of parkin leads to accumulation of PARIS in PD brain cells. PARIS represses the expression of the transcriptional co-activator PGC-1a, which controls the expression of many genes involved in energy metabolism and mitochondrial biogenesis [17]. The parkin-PARIS-PGC-1a pathway is thought to contribute to PD pathogenesis [16,18]. However, the molecular mechanisms controlling the transcriptional activity of PARIS remain largely unclear. In the present study, we showed that PARIS can be SUMOylated and its SUMOylation influences its transcriptional activity. We also showed that PIASy interacts with PARIS and promotes its SUMOylation, whereas it represses the PGC-1a promoter through a PARISindependent mechanism. The present data may improve our understanding of the role of SUMOylation in neurodegeneration in PD. 2. Materials and methods 2.1. Plasmid constructs and siRNA For transient expression of recombinant PARIS proteins in human cell lines, the human PARIS cDNA was amplified by RT-PCR using total mRNA from SH-SY5Y cells, and cloned into pFLAGCMV-2 (SigmaeAldrich, St. Louis, MO), pcDNA3.1/Myc (Invitrogen, Carlsbad, CA) or pM (Clontech, Mountain View, CA), which expresses the GAL4-DNA binding domain (GAL4-DBD) fusion protein. The substitution or deletion mutants of PARIS were created by a PCR-based mutation method using specific mutation primers. SUMO-1, SENP1, and PIAS expression plasmids were described previously [19]. For the reporter assay, the human PGC-1a gene promoter fragment (992 to þ90) was amplified by PCR with the primers 50 -ggggtaccGCTAATAGTGTGTTGGTATTTT-30 and 50 0 TCCTGAATGACGCCAGTCAAGC-3 . This fragment was digested with KpnI/HindIII and then cloned into the KpnI/HindIII site of the pGL3basic vector (designated pGL3-PGC-1a). pL8G5-Luc, pL8-Luc, and pLexA-VP16 were kindly provided by Dr. S. Khochbin [20]. Validated Stealth™ RNAi duplex targeting human PIAS4 (PIASy) and Stealth™ RNAi negative control duplex were purchased from Invitrogen. 2.2. Cell culture and transfection All human cell lines used in this study were maintained in Dulbecco's Modified Eagles' Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. 2.3. Co-immunoprecipitation and immunoblotting Preparation of cell lysates, co-immunoprecipitation, and immunoblotting were performed as described previously [19]. 2.4. Reporter assays Luciferase reporter assays were performed as described previously [19]. Briefly, cells were co-transfected with the reporter construct pRL-CMV, expressing Renilla luciferase, as an internal control, and protein expression constructs, as indicated. At 48 h after transfection, luciferase activities were determined by using a dual luciferase reporter assay system (Promega, Madison, WI) and a GloMax 20/20 luminometer (Promega) according to the manufacturer's instructions. Luciferase activity was normalized to Renilla
luciferase activity from the pRL-CMV vector. The results were presented as the means and standard deviation for relative luciferase activity from at least three independent experiments. 3. Results 3.1. PARIS can be SUMOylated In vivo SUMOylation assays were performed to determine whether PARIS can be SUMOylated in cells. In cells co-transfected with FLAG-PARIS and EGFP-SUMO-1, the FLAG antibody recognized a major 100-kD band (the expected size of the unmodified form of FLAG-PARIS) and two additional slower-migrating 220- and 270-kD bands (Fig. 1A, lane 2). Co-expression of the SUMO-specific protease SENP1 abolished the slower-migrating bands (Fig. 1A, lane 3 and 4), but not the 100-kD band, suggesting that the two slowermigrating bands represent SUMOylated forms of PARIS. PARIS was SUMOylated by SUMO-2/3, but less efficiently than by SUMO-1 in cells (Supplementary Fig. S1). SUMOylation of PARIS was confirmed in vitro (Supplementary Fig. S2). Although SUMO-1 conjugation was more efficient in vivo, the conjugation of SUMO-1 and SUMO-2/3 to PARIS was equally efficient in vitro. 3.2. PARIS is mainly SUMOylated at K189 and K286 SUMOylation typically occurs within the consensus sequence
JKXE, where J represents a large hydrophobic amino acid, K is the lysine conjugated to SUMO, X is any amino acid, and E is a glutamic acid [21]. Analysis of the amino acid sequence of human PARIS revealed that K189 and K286 are located within perfectly matching consensus sequences (Fig. 1B and C). To verify that these lysines were SUMOylation sites, we generated mutants replacing lysine with arginine (KR mutants) or glutamic acid within consensus sequences with alanine (EA mutants), and performed in vivo SUMOylation assays using these mutants (Fig. 1D). Single point mutations (K189R, K286R, E191A or E288A) abolished only the slowest-migrating 270-kD SUMOylated form (lanes 3, 4, 6 and 7). Both double mutants, K189R/K286R (2KR) and E191A/E288A (2EA), completely lost the SUMOylation of PARIS (lanes 5 and 8). The loss of SUMOylation bands in the 2KR mutant was confirmed in vitro (Supplementary Fig. S3). These results indicated that K189 and K286 are the primary SUMOylation sites in the human PARIS protein. 3.3. SUMOylation regulates the transcriptional activity of PARIS PARIS represses the expression of the transcriptional coactivator PGC-1a by binding to insulin response sequences (IRS) in the PGC-1a promoter region in SH-SY5Y cells [16]. We therefore investigated the effect of SUMOylation of PARIS on the PARISmediated repression of PGC-1a promoter activity in cells. For this purpose, reporter assays were performed in four human cell lines co-transfected with pGL3-PGC-1a and wild-type or the 2KR mutant of PARIS (Fig. 2A). In SH-SY5Y cells and H1299 cells, wild-type PARIS repressed the reporter activity as previously reported, and this effect was partially restored by transfection with the 2KR mutant. The 2EA mutant showed almost the same repression activity as the 2KR mutant (data not shown). In HepG2 cells, wild-type PARIS had little effect on reporter activity, whereas cells transfected with the 2KR mutant exhibited significantly higher transcriptional activity than those expressing wild-type PARIS. Conversely, in HEK293 cells, wild-type PARIS activated, whereas 2KR mutant repressed the reporter activity. These results indicated that the effects of PARIS and its SUMOylation on PGC-1a promoter activity are cell type dependent. Next, we investigated the role of the KRAB domain of PARIS in
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
1263
Fig. 1. PARIS is SUMOylated at K189 and K286 in vivo. (A) PARIS can be modified by SUMO-1 in vivo. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS, EGFP-SUMO-1, and Myc-SENP1 [wild-type (WT) or the catalytically inactive mutant C599A (CA)] as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (B) Schematic representation of the human PARIS protein. Its known domains and two lysines (K189 and K286) in the consensus motif for SUMOylation are indicated. (C) Alignment of the sequences surrounding two putative SUMOylation sites (K189 and K286) in the human PARIS protein. (D) K189 and K286 in PARIS are major SUMOylation sites in vivo. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS (WT or the indicated mutants) and EGFP-SUMO-1 as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-FLAG antibody.
Fig. 2. SUMOylation affects the transcriptional activity of PARIS. (A) Effects of SUMOylation of PARIS on PGC-1a promoter activity in different human cell lines. Cells were cotransfected with pGL3-PGC-1a or its control, pGL3-Basic (400 ng), together with expression constructs for FLAG-PARIS (100 ng, WT or the indicated mutants) as indicated. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with empty vector (taken as 1). A schematic representation of the human PGC-1a gene promoterreporter construct is shown at the top. (B) Effects of SUMOylation of PARIS on the L8G5 reporter activity in different human cell lines. Cells were co-transfected with pL8G5-Luc or its control, pL8-Luc (400 ng), together with expression constructs for GAL4-DBD-PARIS (100 ng, WT or the indicated mutants) in the presence or absence of pLexA-VP16 (100 ng) as indicated. The data are expressed relative to the luciferase activity of the pL8G5-Luc co-transfected with control vector pM expressing the GAL4-DBD alone in the presence of pLexAVP16 (taken as 1). A schematic representation of the L8G5-Luc chimeric promoter-reporter construct is shown at the top.
the regulation of PGC-1a promoter activity by generating a KRAB domain deletion mutant (DKRAB). Our results showed that the DKRAB mutant was SUMOylated in cells (data not shown) and
markedly increased reporter activity in all cell lines tested (Fig. 2A). The addition of the 2KR mutation to the DKRAB mutant (DKRAB/ 2KR) reduced the activity to levels similar to those of the 2KR
1264
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
Fig. 3. PIASy enhances the SUMOylation of PARIS in vivo. (A) PIASy enhances the SUMOylation of PARIS by all SUMO isoforms. COS-7 cells were co-transfected with expression constructs for FLAG-PARIS, EGFP-SUMO (SUMO-1, SUMO-2, or SUMO-3) and Myc-PIASy as indicated. The cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (B) PIASy interacts with PARIS in vivo. The cell lysates of COS-7 cells co-expressing FLAG-PARIS (WT or 2KR) and Myc-PIASy WT (top panels), or FLAG-PARIS WT and Myc-PIASy [WT or the C342A mutant (CA)] (bottom panels) as indicated were immunoprecipitated (IP) with anti-FLAG antibody. The precipitates and an aliquot of the cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.
mutant. Similar results were observed with a DKRAB mutant containing a 2EA mutation (data not shown). Although the molecular mechanism remains unclear, these data indicated that SUMOylation of PARIS may be required for its transcriptional activation potential. To examine the function of PARIS in transcription, GAL4-DBDPARIS fusions were constructed and co-transfected with a LexAGal4-driven promoter reporter plasmid (L8G5-Luc) in which the reporter was activated by a fusion protein of the LexA DNA-binding domain and the VP16 activation domain (LexA-VP16) [20]. In the control experiments, L8G5-Luc was substituted by an L8-Luc reporter lacking Gal4-binding sites. These reporters were activated only in the presence of the LexA-VP16 fusion protein (Fig. 2B). In all cell lines tested, wild-type PARIS significantly repressed the LexAVP16-induced reporter activity in a GAL4-binding site-dependent manner, suggesting that PARIS functions as a transcriptional repressor. On the other hand, the 2KR mutant significantly increased the LexA-VP16-induced reporter activity compared with that in controls. In contrast to the reporter assays using the PGC-1a promoter construct, the DKRAB mutation had no effect on the potential activity of PARIS. These results suggested that PARIS has the potential to both activate and repress transcription, and that SUMOylation of PARIS functions in transcriptional repression. SUMOylation of PARIS may switch its role from that of an activator to that of a repressor.
3.4. The SUMO E3 ligase PIASy promotes the SUMOylation of PARIS Next, we searched for the SUMO E3 ligase that specifically SUMOylates PARIS. Several SUMO E3 ligases have been identified to date, and PIAS family members are well known SUMO E3 ligases that target transcription factors [22e24]. Therefore, we examined
the effect of PIAS family proteins on the SUMOylation of PARIS using in vivo SUMOylation assays. Among five PIAS family members analyzed, only PIASy significantly enhanced the SUMOylation of PARIS by all SUMO isoforms and particularly increased the efficiency of SUMO-2/3 conjugation (Supplementary Fig. S4 and Fig. 3A, lanes 3, 5, and 7). To determine whether PIASy interacts with PARIS, COS-7 cells were co-transfected with expression constructs for FLAG-PARIS and Myc-PIASy and analyzed by coimmunoprecipitation. The results showed that PIASy specifically interacted with PARIS (Fig. 3B, lane 3 in each panel), and the 2KR mutation of PARIS or the RING domain mutation, which abolishes the SUMO E3 ligase activity of PIASy, did not affect their interaction (lane 4 in each panel). 3.5. PIASy represses PGC-1a promoter activity PIAS family proteins have both positive and negative effects the activity and expression of many transcription factors in either a SUMOylation-dependent or -independent manner [22e24]. We therefore investigated the effects of PIASy on PGC-1a promoter transcription. SH-SY5Y cells were co-transfected with a PGC-1a promoter reporter together with an expression construct for either wild-type or a SUMO E3 ligase activity-deficient mutant (CA) of PIASy and/or SUMO-1. As shown in Fig. 4A, the expression of wildtype PIASy significantly reduced the reporter activity (lane 4), whereas SUMO-1 alone slightly decreased the activity (lane 3). Coexpression of SUMO-1 with PIASy clearly enhanced the repression of the reporter activity by wild-type PIASy (lane 5). The CA mutant repressed reporter activity less effectively than the wild type PIASy (lane 6), whereas it abolished the enhancing effect of SUMO-1 coexpression on transcriptional repression (lane 7). Conversely, siRNA-mediated knockdown of PIASy in SH-SY5Y cells significantly
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
1265
Fig. 4. Effect of PIASy on PARIS-mediated repression of PGC-1a promoter activity. (A) PIASy represses PGC-1a promoter activity. SH-SY5Y cells were co-transfected with pGL3PGC-1a or its control, pGL3-basic (400 ng), together with expression constructs for FLAG-PIASy [100 ng, WT or C342A (CA)] and EGFP-SUMO-1 (100 ng) as indicated. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with empty vectors (taken as 1). (B) Effects of PIASy knockdown on PGC-1a promoter activity. SH-SY5Y cells were co-transfected with pGL3-PGC-1a (400 ng), together with negative control siRNA (40 pmol), PIASy-siRNA (40 pmol) or vehicle (non-siRNA control) as indicated. The data are expressed relative to the luciferase activity in the presence of negative control siRNA duplexes (taken as 1). Immunoblotting of cell lysates from the luciferase assay was performed to confirm the knockdown of PIASy proteins. b-actin was used as the loading control (bottom panels). (C) PIASy attenuates the PARIS-mediated repression of PGC-1a promoter activity in a SUMOylation status-dependent manner. SH-SY5Y cells were co-transfected with pGL3-PGC-1a or its control, pGL3-basic (400 ng), together with expression constructs for FLAG-PIASy WT (100 ng), FLAG-PARIS (100 ng, either WT or 2KR) and EGFP-SUMO-1 (100 ng) as indicated. Cells were incubated for 48 h and analyzed for luciferase activity. The data are expressed relative to the luciferase activity of pGL3-PGC-1a co-transfected with pRL-CMV and empty vectors (taken as 1). (D) PIASy-mediated repression of PGC-1a promoter activity is independent of HDAC activity. SH-SY5Y cells were co-transfected with pGL3-PGC-1a (400 ng), in the absence or presence of expression constructs for FLAG-PARIS (100 ng) and/or FLAG-PIASy (100 ng) as indicated. At 24 h after the transfection, cells were treated with increasing doses of trichostatin A (TSA) for an additional 24 h and then analyzed for luciferase activity. The data are expressed relative to the luciferase activity in the absence of TSA (taken as 1).
increased the reporter activity (Fig. 4B, lane 3). These results suggested that PIASy is involved in the repression of the PGC-1a promoter in either a SUMOylation-dependent or -independent manner. 3.6. PIASy influences PARIS-mediated repression of PGC-1a promoter activity Next, we examined the effects of expression of PIASy on the PARIS-mediated repression of PGC-1a promoter activity in SH-SY5Y cells (Fig. 4C). Co-transfection with the PGC-1a promoter reporter together with an expression construct for either PIASy or wild-type PARIS significantly decreased reporter activity (lanes 4 and 6). However, co-expression of PIASy with wild-type PARIS, but not with the 2KR mutant, attenuated its repressive activity (lanes 8 and 12). By contrast, co-expression of PIASy with the 2KR mutant synergistically repressed the reporter activity, although the 2KR mutant alone did not repress the reporter activity. Co-expression
SUMO-1 with PIASy completely repressed the reporter activity independently of PARIS (lanes 5, 9, and 13). These results indicated that PIASy affects the PARIS-mediated repression of PGC-1a promoter activity in a manner dependent on the SUMOylation status of PARIS, and that PIASy-mediated repression predominates over PARIS activity in SUMO-1 overexpressing cells. 3.7. PIASy-mediated repression of PGC-1a promoter activity is independent of HDAC activity PIASy represses the activity of several transcription factors and co-factors by recruiting HDACs [25,26]. Therefore, we examined whether HDAC activity is required for PIASy-mediated repression of PGC-1a promoter activity. SH-SY5Y cells were co-transfected with a PGC-1a promoter reporter together with or without PIASy and/or PARIS, and treated with increasing amounts of trichostatin A (TSA), an HDAC inhibitor, followed by determination of reporter activities. As shown in Fig. 4D, treatment with increasing doses of TSA
1266
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267
increased PGC-1a promoter activity in control and PARIS alone transfected cells (top and second panels). However, TSA had almost no effect on PIASy-mediated repression of PGC-1a promoter activity in the absence and presence of PARIS (third and bottom panels). These results suggested that PARIS-mediated repression, but not PIASy-mediated repression, depends on HDAC activity, and that PIASy does not recruit a repressor complex with HDAC activity to the PGC-1a promoter. 4. Discussion In the present study, we demonstrated that PARIS can be modified by SUMO, and that its SUMOylation plays an important role in the regulation of its transcriptional activity. A SUMOylation deficient mutant restored the repression of PGC-1a promoter activity in SH-SY5Y cells, suggesting that the SUMOylation of PARIS plays a role in its repressive activity. However, our results suggested that the PARIS-mediated regulation of PGC-1a promoter activity is more complex. Reporter analyses showed that the effect of PARIS on the PGC-1a promoter varied significantly in a cell type dependent manner. The fact that the PGC-1a promoter fragment (992 to þ90) contains several consensus transcription factor binding sites, including GC-box, CRE, IRS, SRE, and E-Box [27], indicates that PARIS may interact with other transcription factors that bind to these sites on the promoter. Moreover, co-factors binding to PARIS and/or other transcription factors may affect the ability of PARIS to regulate the PGC-1a promoter activity. The effect of PARIS on the PGC-1a promoter could be affected by basal levels of expression of other transcription factors and/or cofactors in different cell types. Similar to other KRAB-ZFPs, the KRAB domain of PARIS played an important role in its transcriptional repression activity. The DKRAB mutant enhanced PGC-1a promoter activity, whereas it had no effect on LexA-VP16-induced promoter activity, suggesting that the KRAB-mediated interaction of PARIS with co-factor(s) plays a role in PGC-1a promoter repression. KAP-1 is an essential co-factor of KRAB-ZFPs [28]. The interaction of KAP-1 with KRAB-ZNFs promotes the recruitment of a repression complex [9e11]. We investigated the interaction of KAP-1 with PARIS by co-immunoprecipitation assays and its effect on PARIS-mediated transcriptional repression of the PGC-1a promoter was analyzed using reporter assays. An interaction between KAP-1 and PARIS was not detected in the present study. Moreover, KAP-1 overexpression or knockdown in SH-SY5Y cells did not significantly affect the PARIS-mediated transcriptional repression (data not shown). However, we cannot completely rule out the possibility that KAP-1 is involved in the repressive activity of PARIS. Our results showed that PIASy acts as a SUMO E3 ligase for PARIS and is involved in the repression of the PGC-1a promoter in SH-SY5Y cells. Expression of PIASy alone repressed PGC-1a promoter activity in the absence of PARIS (Fig. 4A). Co-expression of PIASy and PARIS significantly attenuated their mutually repressive effect, although this attenuation was compromised by the SUMOylation-deficient mutation of PARIS or co-expression of SUMO-1 (Fig. 4C). The PIASy repression of PGC-1a promoter activity occurred through an HDAC-independent mechanism, whereas the repressive activity of PARIS markedly depended on HDAC activity (Fig. 4D). Taken together, these findings suggest that PIASy-mediated SUMOylation and interaction do not contribute to the repressive activity of PARIS. However, the relatively modest and transient SUMOylation of PARIS may be necessary to attenuate the repression of the PGC-1a promoter by co-expression of PIASy. Further studies are needed to improve our understanding of the molecular mechanisms underlying the PARIS-mediated regulation of PGC-1a promoter activity.
Conflicts of interest The authors declare no conflicts of interest. Acknowledgements We thank Dr. Saadi Khochbin (University Grenoble-Alpes, France) for providing reporter plasmids, and Dr. Shinji Oikawa (Mie University, Japan) for providing SH-SY5Y cells. This work was supported in part by Grants-in-Aid for the Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant Numbers 24500406 and 15K06738 to T.N.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.04.051. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.051. References [1] R. Urrutia, KRAB-containing zinc-finger repressor proteins, Genome Biol. 4 (2003) 231e238. [2] S. Huntley, D.M. Baggott, A.T. Hamilton, M. Tran-Gyamfi, S. Yang, J. Kim, L. Gordon, E. Branscomb, L. Stubbs, A comprehensive catalog of human KRABassociated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors, Genome Res. 16 (2006) 669e677. [3] A. Lupo, E. Cesaro, G. Montano, D. Zurlo, P. Izzo, P. Costanzo, KRAB-Zinc finger proteins: a repressor family displaying multiple biological functions, Curr. Genomics 14 (2013) 268e278. [4] J.F. Margolin, J.R. Friedman, W.K. Meyer, H. Vissing, H.J. Thiesen, F.J. Rauscher 3rd, Krüppel-associated boxes are potent transcriptional repression domains, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4509e4513. [5] R. Witzgall, E. O'Leary, A. Leaf, D. Onaldi, J.V. Bonventre, The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4514e4518. [6] J.R. Friedman, W.J. Fredericks, D.E. Jensen, D.W. Speicher, X.P. Huang, E.G. Neilson, F.J. Rauscher 3rd, KAP-1, a novel corepressor for the highly conserved KRAB repression domain, Genes Dev. 10 (1996) 2067e2078. [7] P. Moosmann, O. Georgiev, B. Le Douarin, J.P. Bourquin, W. Schaffner, Transcriptional repression by RING finger protein TIF1b that interacts with the KRAB repressor domain of KOX1, Nucleic Acids Res. 24 (1996) 4859e4867. [8] S.S. Kim, Y.M. Chen, E. O'Leary, R. Witzgall, M. Vidal, J.V. Bonventre, A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15299e15304. [9] D.C. Schultz, J.R. Friedman, F.J. Rauscher III, Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD, Genes Dev. 15 (2001) 428e443. http://www.ncbi.nlm.nih. gov/pubmed/11230151. comments. [10] D.C. Schultz, K. Ayyanathan, D. Negorev, G.G. Maul, F.J. Rauscher 3rd, SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zincfinger proteins, Genes Dev. 16 (2002) 919e932. http://www.ncbi.nlm.nih. gov/pubmed/11959841. comments. [11] S.P. Sripathy, J. Stevens, D.C. Schultz, The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression, Mol. Cell. Biol. 26 (2006) 8623e8638. [12] F. Melchior, SUMO-nonclassical ubiquitin, Annu. Rev. Cell Dev. Biol. 16 (2000) 591e626. [13] R.T. Hay, SUMO: a history of modification, Mol. Cell 18 (2005) 1e12. [14] K.D. Sarge, O.K. Park-Sarge, Sumoylation and human disease pathogenesis, Trends. Biochem. Sci. 34 (2009) 200e205. [15] D. Mukhopadhyay, M. Dasso, Modification in reverse: the SUMO proteases, Trends. Biochem. Sci. 32 (2007) 286e295. [16] J.H. Shin, H.S. Ko, H. Kang, Y. Lee, Y.I. Lee, O. Pletinkova, J.C. Troconso, V.L. Dawson, T.M. Dawson, PARIS (ZNF746) repression of PGC-1a contributes to neurodegeneration in Parkinson's disease, Cell 144 (2011) 689e702. [17] J.A. Villena, New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond, FEBS J. 282 (2015) 647e672.
T. Nishida, Y. Yamada / Biochemical and Biophysical Research Communications 473 (2016) 1261e1267 [18] D.A. Stevens, Y. Lee, H.C. Kang, B.D. Lee, Y.I. Lee, A. Bower, H. Jiang, S.U. Kang, S.A. Andrabi, V.L. Dawson, J.H. Shin, T.M. Dawson, Parkin loss leads to PARISdependent declines in mitochondrial mass and respiration, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 11696e11701. [19] T. Nishida, M. Terashima, K. Fukami, PIASy-mediated repression of the Ets-1 is independent of its sumoylation, Biochem. Biophys. Res. Commun. 345 (2006) 1536e1546. [20] C. Lemercier, A. Verdel, B. Galloo, S. Curtet, M.P. Brocard, S. Khochbin, mHDA1/ HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity, J. Biol. Chem. 275 (2000) 15594e15599. [21] M.S. Rodriguez, C. Dargemont, R.T. Hay, SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting, J. Biol. Chem. 276 (2001) 12654e12659. [22] D. Schmidt, S. Müller, PIAS/SUMO: new partners in transcriptional regulation, Cell. Mol. Life Sci. 60 (2003) 2561e2574. [23] A.D. Sharrocks, PIAS proteins and transcriptional regulation-more than just SUMO E3 ligases? Genes Dev. 20 (2006) 754e758.
1267
€a €skel€ [24] M.M. Rytinki, S. Kaikkonen, P. Pehkonen, T. Ja ainen, J.J. Palvimo, PIAS proteins: pleiotropic interactors associated with SUMO, Cell. Mol. Life Sci. 66 (2009) 3029e3041. [25] J. Long, I. Matsuura, D. He, G. Wang, K. Shuai, F. Liu, Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 9791e9796. [26] M. Gross, R. Yang, I. Top, C. Gasper, K. Shuai, PIASy-mediated repression of the androgen receptor is independent of sumoylation, Oncogene 23 (2004) 3059e3066. [27] I. Irrcher, V. Ljubicic, A.F. Kirwan, D.A. Hood, AMP-activated protein kinaseregulated activation of the PGC-1a promoter in skeletal muscle cells, PloS One 3 (2008) e3614. [28] M. Abrink, J.A. Ortiz, C. Mark, C. Sanchez, C. Looman, L. Hellman, P. Chambon, R. Losson, Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 b, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1422e1426.