The UPR (unfolded protein response), a cellular defence mechanism against misfolded protein accumulation in the ER (endoplasmic reticulum), is associated with many human diseases such as aging, cancer and diabetes. XBP1 (X-box-binding protein 1), a key transcription factor of the UPR, is critical in maintaining ER homoeostasis. Nevertheless, the mechanism by which XBP1 transcriptional activity is regulated remains unexplored. In the present study we show that XBP1s, the active spliced form of XBP1 protein, is SUMOylated, mainly by PIAS2 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 2] at two lysine residues located in the C-terminal transactivation domain. Ablation of these SUMOylation events significantly enhances the transcriptional activity of XBP1s towards UPR target genes. Thus our results reveal an unexpected role for SUMO (small ubiquitin-related modifier) in the regulation of UPR activation and ER homoeostasis.

INTRODUCTION

The IRE1α (inositol-requiring enzyme 1α)/XBP1 (X-box-binding protein 1) pathway, the most highly conserved signalling branch of the UPR (unfolded protein response) [13], is important for ER (endoplasmic reticulum) biogenesis and the secretory capacity of cells. Once activated, mammalian IRE1α splices 26 nucleotides from the Xbp1u (unspliced XBP1) mRNA, leading to a frameshift and the generation of an active bZIP (basic leucine zipper) transcription factor called XBP1s (spliced XBP1) that contains a C-terminal transactivation domain absent from XBP1u [49]. The XBP1s protein subsequently translocates to the nucleus and activates the transcription of genes involved in lipid biosynthesis, protein folding, degradation and trafficking [6,7,10,11].

Studies in animal models have revealed that the IRE1α/XBP1 pathway is essential for the development and function of the liver and heart, as well as ‘professional’ secretory cells, such as pancreatic and plasma cells [1216]. XBP1s has also been shown to regulate hepatic lipogenesis in an UPR-independent manner [17]. We showed recently that the IRE1α/XBP1 pathway is indispensable for adipocyte differentiation via XBP1s-mediated regulation of a key adipogenic factor C/EBPα (CCAAT/enhancer-binding protein α) [18]. Despite its physiological significance, the mechanism by which the transcriptional activity of XBP1s protein is regulated remained largely unknown.

The post-translational modification of proteins by SUMO (small ubiquitin-like modifier) is an important transient regulatory mechanism in many cellular processes, most notably transcriptional regulation, DNA damage and signal transduction [19,20]. SUMO proteins have three isoforms (SUMO1–3), with 50% identity between SUMO1 and SUMO2/3. Similar to the process of ubiquitination, SUMO is first activated by the E1 enzyme Sae1 (SUMO1-activating enzyme subunit 1)–Uba2 (ubiquitin-like modifier activating enzyme 2), then is transferred on to the E2 enzyme Ubc9 and subsequently conjugated to target proteins via the activity of an E3 ligase, such as PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)], RanBP2 (Ran-binding protein 2) or the polycomb group protein Pc2 [19,20]. In the present study we show that the transcriptional activity of XBP1s is negatively modulated by SUMOylation. Our results reveal an unexpected role for SUMO in the regulation of UPR activation.

EXPERIMENTAL

Plasmids

Full-length expression constructs for XBP1s, XBP1u, ATF6 (activating transcription factor 6) and the ATF6 active form ATF6N (nuclear ATF6) were cloned from a liver cDNA library and subcloned into either pcDNA3/FLAG or pcDNA3/HA or pcDNA3/Myc vectors to tag the proteins. The truncated XBP1s encodes amino acids 1–260 of full-length XBP1s. XBP1s–GFP (green fluorescent protein) fusion constructs were made by subcloning the XBP1s PCR fragment into the pEGFP-N1 vector (Clontech). To construct the plasmid encoding the luciferase gene driven by four XBP1-binding sites, a synthetic oligonucleotide containing four XBP1-binding sites, 5′-aagctagccgcgTGGATGACGTGTACATGGATGACGTGTACATGGATGACGTGTACATGGATGACGTGTACAaagctttt-3′ (italicized sequences represent the XBP1-binding sites; lowercase sequences were added to provide restriction enzyme sites for NheI and HindIII, which are underlined), was digested with NheI/Hind III and then ligated into the NheI/HindIII site of the pGL3 vector (Promega). mPIAS1/2 (mouse PIAS1/2) was cloned from a mouse liver cDNA library template and subcloned into the pcDNA3/FLAG vector using the NotI and XbaI sites. Plasmids encoding hPIAS2a (human PIAS2a) and hPIAS2b were gifts from Jorma Palvimo (University of Kuopio, Kuopio, Finland). Plasmids encoding FLAG-tagged HDAC (histone deacetylase) 1, 2, 4, 5 and 6 in the pBJ5 vector, FLAG-tagged HDAC3 in the pcDNA3 vector and the β-galactosidase expression plasmid were gifts from Marc Montminy (Salk Institute, La Jolla, CA, U.S.A.). The FLAG–hPIAS4-, Myc–Ubc9- and HA (haemagglutinin)–SUMO1-expressing plasmids were gifts from Ling Cai (University of California San Diego, La Jolla, CA, U.S.A.). HA–SUMO2- and HA–SUMO3-expressing plasmids were gifts from Katherine Kieckhafer (Department of Molecular Biology and Genetics, Cornell University, New York, U.S.A.). The same amount of plasmids was used in all transfection experiments.

Cells and reagents

XBP1−/− MEFs (mouse embryonic fibroblasts; a gift from Laurie Glimcher, Harvard Medical School, Boston, MA, U.S.A.), macrophage RAW264.7 and HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS (fetal bovine serum; Hyclone). The ER stress inducer Tg (thapsigargin; EMD Calbiochem) was dissolved in DMSO and used at 300 nM.

Site-directed mutagenesis

Site-directed mutagenesis to generate XBP1s mutants was performed using pcDNA3/FLAG–XBP1s as the template. The PCR included the mutagenic primers, as well as the Pfu polymerase (Stratagene) and the program consisted of 13 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 5 min. After DpnI digestion for 2–3 h, the PCR product was transformed into XL-Blue competent cells by incubating plasmids or PCR products with the cells for 30 min on ice, followed by a heat-shock at 42 °C for 30 s. The correct introduction of the mutants was confirmed by DNA sequencing. All primers used in mutagenesis are listed in Supplementary Table S1 (at http://www.BiochemJ.org/bj/429/bj4290095add.htm).

Fluorescent microscopy

HEK-293T cells were placed on to sterile cover slips coated with poly-L-lysine (Sigma–Aldrich). Cells were transfected with plasmids encoding the wild-type or mutant XBP1s–GFP fusion proteins. Cover slips were removed from dishes and mounted on to slides with Vectashield® and DAPI (4′,6-diamidino-2-phenylindole). The images were taken on a Leica DM5000 B microscope using a 63× oil objective.

Immunoprecipitation

HEK-293T cells were plated on to 10-cm-diameter dishes the day before transfection. Transfection was performed by preparing PEI (polyethylenimiene) (1 mg/ml in 0.9% NaCl) with plasmids at a 4:1 (μl/μg) ratio and adding to the cell culture. Transfected cells were scrapped off the plates with cold lysis buffer [50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, 0.5 mM DTT (dithiothreitol) and protease inhibitor cocktail], containing 10 mM N-ethylmaleimide (Sigma–Aldrich) when detecting SUMOylation, and sonicated with a Branson digital sonifier 25 for 10 s. Lysate were incubated on ice for 15 min and centrifuged at 16000 g for 15 min. Anti-FLAG–agarose beads (Sigma–Aldrich) were added to the supernatants and rocked gently at 4 °C overnight. Following a wash with lysis buffer and two washes with wash buffer [20 mM Tris/HCl, pH7.5, 137 mM NaCl, 1% (v/v) Triton X-100, 2 mM EDTA, 10% (v/v) glycerol and 0.5 mM DTT], the beads were boiled and subjected to centrifugation (300 g for 1 min). Supernatants were analysed by SDS/PAGE (8% gels) and Western blotting.

Western blotting

Western blotting was performed using 15–30 μg of total cell lysates as described in [18]. The antibodies were anti-FLAG antibody and anti-HA–HRP (horseradish peroxidase) antibody (both Sigma–Aldrich), rabbit anti-XBP1 antibody (Santa Cruz Biotechnology) and goat anti-(rabbit IgG)–HRP (BioRad).

Luciferase reporter assay

HEK-293T cells and XBP1−/− MEF cells were transfected on to 24-well for 24 h via PEI and Lipofectamine™ 2000 respectively. Cells were lysed in 100 μl of extraction buffer [Gly-Gly buffer (Fisher Scientific), 1% (v/v) Triton X-100 and 1 mM DTT]. Lysates (50 μl) were transferred on to a 96-well plate containing 50 μl of assay mix (Gly-Gly buffer, 16 mM K2HPO4, 2 mM DTT and 2.5 mM ATP). Luciferin mix (50 μl of a 0.4 mM solution; Xenogen) was added to each well through an injector and readings were collected using the Synergy 2 plate reader (BioTek). Luciferase activity was normalized to activity from a co-transfected CMV (cytomegalovirus)-promoter-driven β-galactosidase expression plasmid.

RNA extraction and qRT-PCR (quantitative real-time PCR)

Total RNA was extracted using TRIzol® according to the supplier's protocol (Molecular Research Center) and reverse transcribed using the Superscript III kit (Invitrogen). cDNAs were analysed using the SYBR Green PCR system on the Roche LightCycler 480 machine. All qRT-PCR results were normalized to the level of the ribosomal l32 gene. Primer sequences are listed in Supplementary Table S1.

Statistical analysis

Results are expressed as means±S.E.M. Comparisons between groups were made by unpaired two-tailed Student's t tests. P<0.05 was considered as statistically significant. All experiments were repeated at least twice and representative results are shown.

RESULTS

XBP1s of the UPR is SUMOylated

To identify potential UPR transcription factors that could be SUMOylated, we transfected HEK-293T cells with vectors encoding XBP1s, ATF6 and ATF6N, plus the E2 enzyme Ubc9 and SUMO1. Among the three factors, XBP1s protein was the only one which underwent SUMOylation (Figure 1A). To confirm further the specificity of SUMOylation in the UPR, we found that XBP1s, but not XBP1u, was SUMOylated by all three isoforms of SUMO, i.e. SUMO1 and SUMO2/3 (Figure 1B). Indeed, in a recent genome-wide analysis of SUMO2 modifications during the heat-shock response, XBP1s was identified as a protein that was SUMOylated by SUMO2 [21]. In line with previous reports [2224], SUMOylation increased the molecular mass of XBP1s protein by approx. 20 kDa. XBP1s SUMOylation was not cell-type specific as it was also detected in MEFs and COS7 cells (results not shown). Taken together, our results show that the XBP1s protein of the UPR is SUMOylated.

XBP1s is SUMOylated

Figure 1
XBP1s is SUMOylated

Immunoblots of HA–SUMO showing recovery of HA–SUMO from immunoprecipitates (IP) with anti-FLAG antibody. (A) HEK-293T cells were transfected with full-length (FL) FLAG-tagged XBP1s, ATF6 or ATF6N together with HA–SUMO1. Two SUMOlyated XBP1s variants were indentified, denoted by sumon (arrows). Inputs are shown in the middle and bottom panels. *, non-specific band. (B) HEK-293T cells were transfected with FLAG–XBP1s or FLAG–XBP1u, together with different SUMO isoforms (HA–SUMO1, 2 or 3). Only XBP1s is SUMOylated. The molecular mass in kDa is indicated on the left-hand side.

Figure 1
XBP1s is SUMOylated

Immunoblots of HA–SUMO showing recovery of HA–SUMO from immunoprecipitates (IP) with anti-FLAG antibody. (A) HEK-293T cells were transfected with full-length (FL) FLAG-tagged XBP1s, ATF6 or ATF6N together with HA–SUMO1. Two SUMOlyated XBP1s variants were indentified, denoted by sumon (arrows). Inputs are shown in the middle and bottom panels. *, non-specific band. (B) HEK-293T cells were transfected with FLAG–XBP1s or FLAG–XBP1u, together with different SUMO isoforms (HA–SUMO1, 2 or 3). Only XBP1s is SUMOylated. The molecular mass in kDa is indicated on the left-hand side.

XBP1s is SUMOylated at lysine residues in the C-terminal transactivation domain

SUMOylation often occurs at the lysine residues in the motif ΨKXE/D (where Ψ is a hydrophobic residue, X is any residue and E/D is aspartate/glutamate) [25,26]. Two highly conserved SUMOylation motifs were identified at residues Lys276 and Lys297 within the transactivation domain of the XBP1s protein, a region that is absent from the XBP1u protein (Figure 2A). We performed site-directed mutagenesis to target these residues and found that introducing a K276R mutation in XBP1s abolished one SUMOylation event (Figure 2B). Interestingly, the K297R mutation completely abolished SUMOylation on XBP1s, suggesting that SUMOylation at Lys297 is required for SUMOylation at Lys276. To exclude the possibility of non-specific modification events, we determined that the K298R mutation, i.e. the X in the ΨKXE motif, had no effect on the SUMOylation of the XBP1s protein (Figure 2B). Thus our results show that the XBP1s protein is SUMOylated at two lysine residues located in the C-terminal transactivation domain.

XBP1s is SUMOylated at Lys276 and Lys297 in the C-terminal transactivation domain

Figure 2
XBP1s is SUMOylated at Lys276 and Lys297 in the C-terminal transactivation domain

(A) A schematic diagram of mouse XBP1u and XBP1s with shared regions shown with the same colour (DBD, DNA-binding domain; TAD, transactivation domain). The conservation of two putative SUMOylation motifs in XBP1s is shown in the lower panel. (B) Diagram of the mutagenesis of lysine residues (K) to arginine residues (R) at the SUMOylation consensus motifs of mouse XBP1s protein (top panel). Mutated residues are underlined. The middle panel shows immunoblots with anti-HA antibody conjugated to HRP (HA–HRP), to detect SUMO1, from immunoprecipitates (IP) of different FLAG-tagged XBP1s constructs in HEK-293T cells upon transfection with or without HA–SUMO1. The input of XBP1s is shown in the bottom panel. The same amounts of plasmids were added in all experiments and the reason for the variation between samples remains unclear. The molecular mass in kDa is indicated on the left-hand side. *, non-specific band; FL, full-length; sumon, SUMOylated variant n.

Figure 2
XBP1s is SUMOylated at Lys276 and Lys297 in the C-terminal transactivation domain

(A) A schematic diagram of mouse XBP1u and XBP1s with shared regions shown with the same colour (DBD, DNA-binding domain; TAD, transactivation domain). The conservation of two putative SUMOylation motifs in XBP1s is shown in the lower panel. (B) Diagram of the mutagenesis of lysine residues (K) to arginine residues (R) at the SUMOylation consensus motifs of mouse XBP1s protein (top panel). Mutated residues are underlined. The middle panel shows immunoblots with anti-HA antibody conjugated to HRP (HA–HRP), to detect SUMO1, from immunoprecipitates (IP) of different FLAG-tagged XBP1s constructs in HEK-293T cells upon transfection with or without HA–SUMO1. The input of XBP1s is shown in the bottom panel. The same amounts of plasmids were added in all experiments and the reason for the variation between samples remains unclear. The molecular mass in kDa is indicated on the left-hand side. *, non-specific band; FL, full-length; sumon, SUMOylated variant n.

XBP1s protein is SUMOylated endogenously

To determine whether the endogenous SUMOylation machinery could mediate SUMOylation of XBP1s, cells were transfected with various XBP1s plasmids in the absence of exogenous Ubc9 and SUMO1. Indeed, both wild-type and K298R XBP1s were SUMOylated; the K276R mutant was modified to a lesser extent and no SUMOylation occurred in the K297R mutant or K276R/K297R double-mutant (Figure 3A). The differences between the XBP1 and SUMO blots were highly reproducible and specific; therefore we speculated that this was probably because the anti-SUMO antibody preferentially recognizes the bi-SUMOylated protein (Figure 3A). Furthermore, SUMOylated endogenous XBP1s protein could be detected following immunoprecipitation of XBP1 in macrophage cells treated with the ER stress inducer Tg (Figure 3B). Nonetheless, ER stress itself did not increase the SUMOylation of XBP1s as Tg treatment failed to alter the amount of SUMO–XBP1s in HEK-293T cells transfected with XBP1s (Figure 3C). Thus our results suggest that the XBP1s protein may be constitutively SUMOylated during ER stress.

Endogenous SUMOylation of XBP1s

Figure 3
Endogenous SUMOylation of XBP1s

(A) Immunoblots with anti-XBP1 antibody (upper panel) and anti-SUMO1 antibody (lower panel) from immunoprecipitates (IP) of anti-FLAG–agarose prepared from HEK-293T cells transfected with different FLAG–XBP1s constructs without exogenous Ubc9 or SUMO1 [in the right-hand most lane where wild-type (wt) XBP1s, E2 Ubc9 and SUMO1 were included as a positive control]. Note that exogenous SUMO is HA-tagged, thus larger than endogenous SUMO. (B) Immunoblots of SUMO1 (upper panel) and XBP1 (lower panel) from immunoprecipitates (IP) of XBP1 prepared from RAW264.7 macrophage cells treated with 300 nM Tg for 4 h. Immunoprecipitates of rabbit IgG were included as a negative control. (C) Immunoblots with anti-FLAG antibody conjugated to HRP (upper panel) and anti-XBP1s antibody (lower panel) from immunoprecipitates with anti-FLAG–agarose prepared from FLAG–XBP1s- and SUMO-transfected HEK-293T cells. Cells were treated with 300 nM Tg for 0, 1, 2 or 3 h as indicated prior to immunoprecipitation. The molecular mass in kDa is indicated on the left-hand side. *, non-specific bands; FL, full-length; sumon, SUMOylated variant n.

Figure 3
Endogenous SUMOylation of XBP1s

(A) Immunoblots with anti-XBP1 antibody (upper panel) and anti-SUMO1 antibody (lower panel) from immunoprecipitates (IP) of anti-FLAG–agarose prepared from HEK-293T cells transfected with different FLAG–XBP1s constructs without exogenous Ubc9 or SUMO1 [in the right-hand most lane where wild-type (wt) XBP1s, E2 Ubc9 and SUMO1 were included as a positive control]. Note that exogenous SUMO is HA-tagged, thus larger than endogenous SUMO. (B) Immunoblots of SUMO1 (upper panel) and XBP1 (lower panel) from immunoprecipitates (IP) of XBP1 prepared from RAW264.7 macrophage cells treated with 300 nM Tg for 4 h. Immunoprecipitates of rabbit IgG were included as a negative control. (C) Immunoblots with anti-FLAG antibody conjugated to HRP (upper panel) and anti-XBP1s antibody (lower panel) from immunoprecipitates with anti-FLAG–agarose prepared from FLAG–XBP1s- and SUMO-transfected HEK-293T cells. Cells were treated with 300 nM Tg for 0, 1, 2 or 3 h as indicated prior to immunoprecipitation. The molecular mass in kDa is indicated on the left-hand side. *, non-specific bands; FL, full-length; sumon, SUMOylated variant n.

SUMO E3 ligase PIAS2 interacts with and mediates SUMOylation of XBP1s

To identify the E3 ligase responsible for SUMOylation of XBP1s, we transfected HEK-293T cells with XBP1s and various E3 ligase PIAS constructs (PIAS1, 2a/b, 3 and 4). Strikingly, XBP1s protein interacted specifically with PIAS2 proteins, either mPIAS2 or its human homologue hPIAS2b. Interactions with other PIAS proteins, including PIAS1, 3, and 4 were either much weaker or not detected (Figure 4A and results not shown). Similar observations were observed for XBP1u (Figure 4B), suggesting that the N-terminal region, containing the DNA-binding domain shared by both XBP1u and XBP1s (Figure 2A), may mediate the interaction between XBP1s and PIAS2. The interaction of XBP1s or XBP1u protein with hPIAS2a was much weaker compared with that for mPIAS2 or hPIAS2b (Figures 4A and 4B). It is worth pointing out that the same amount of plasmids for XBP1s was used in all experiments and the difference in the level of XBP1 protein (Figures 4A and 4B) was probably due to the stabilization of XBP1s proteins by the interacting proteins.

SUMO E3 ligase PIAS2 interacts with and increases SUMOylation of XBP1s

Figure 4
SUMO E3 ligase PIAS2 interacts with and increases SUMOylation of XBP1s

(A and B) Immunoblots showing recovery of (A) HA–XBP1s and (B) HA–XBP1u from immunoprecipitates (IP) of FLAG-tagged PIAS proteins prepared from transfected HEK-293T cells. *, non-specific bands. (C) Domain comparison of PIAS proteins and the identity shared with mPIAS2 protein. SAP, scaffold attachment factor A/B, acinus and PIAS; RING, RING zinc finger; SIM, SUMO-interacting motif; S/T, serine/threonine-rich domain. Numbers indicate the position of residues. (D) Immunoblots of XBP1 from immunoprecipitates (IP) of FLAG in HEK-293T cells transfected with wild-type (wt) or mutant FLAG-tagged XBP1, and FLAG–hPIAS2b and/or E2 Myc–Ubc9. Note that the same amounts of plasmids were used in each condition in all experiments. The molecular mass in kDa is indicated on the left-hand side for (A, B and D) FL, full-length; sumon, SUMOylated variant n.

Figure 4
SUMO E3 ligase PIAS2 interacts with and increases SUMOylation of XBP1s

(A and B) Immunoblots showing recovery of (A) HA–XBP1s and (B) HA–XBP1u from immunoprecipitates (IP) of FLAG-tagged PIAS proteins prepared from transfected HEK-293T cells. *, non-specific bands. (C) Domain comparison of PIAS proteins and the identity shared with mPIAS2 protein. SAP, scaffold attachment factor A/B, acinus and PIAS; RING, RING zinc finger; SIM, SUMO-interacting motif; S/T, serine/threonine-rich domain. Numbers indicate the position of residues. (D) Immunoblots of XBP1 from immunoprecipitates (IP) of FLAG in HEK-293T cells transfected with wild-type (wt) or mutant FLAG-tagged XBP1, and FLAG–hPIAS2b and/or E2 Myc–Ubc9. Note that the same amounts of plasmids were used in each condition in all experiments. The molecular mass in kDa is indicated on the left-hand side for (A, B and D) FL, full-length; sumon, SUMOylated variant n.

As splicing variants of PIAS2a (PIASxα) and PIAS2b (PIASxβ) differ from each other at the C-terminal serine/threonine (S/T)-rich domain (Figure 4C), our results suggest that this domain of the PIAS protein may partially mediate the interaction with XBP1s. Furthermore, and supporting its role in SUMOylation of XBP1s protein, overexpression of hPIAS2b increased SUMOylation of wild-type but not K276R/K297R XBP1s proteins (Figure 4D). Thus our results suggest that mPIAS2 or hPIAS2b interacts with and mediates SUMOylation of XBP1s.

SUMOylation does not alter the nuclear localization of XBP1s

SUMOylation of transcription factors often leads to alterations in the intracellular localization of target proteins [19]. To address this, we expressed wild-type or mutant XBP1s–GFP fusion proteins in HEK-293T cells. As with wild-type XBP1s proteins, SUMOylation mutant proteins predominantly localized to the nucleus and did not exhibit altered intracellular distribution (Figure 5A). Thus our results suggest that SUMOylation does not affect the nuclear localization of XBP1s protein.

SUMOylation of XBP1s does not affect its nuclear localization nor its interaction with HDAC5

Figure 5
SUMOylation of XBP1s does not affect its nuclear localization nor its interaction with HDAC5

(A) Fluorescent microscopic images of XBP1s–GFP fusion constructs upon 24 h of transfection in HEK-293T cells. Nuclei were counter-stained with DAPI. wt, wild-type. (B) Immunoblots of XBP1 from immunoprecipitates (IP) with anti-FLAG–agarose prepared from HEK-293T cells transfected with Myc-tagged XBP1s with or without different FLAG–HDAC constructs (HDAC1–6). Note that same amount of plasmids were transfected in each condition. Inputs are shown in the lower two panels. (C) Immunoblots of XBP1 from immunoprecipitates (IP) with anti-FLAG–agarose prepared from HEK-293T cells transfected with FLAG–HDAC5 together with different HA–XBP1s constructs (left-hand panel) or Myc–XBP1s constructs (right-hand panel). Inputs for XBP1s and HDAC5 are shown (lower panels). s, XBP1; *, non-specific band. The molecular mass in kDa is indicated on the left-hand side.

Figure 5
SUMOylation of XBP1s does not affect its nuclear localization nor its interaction with HDAC5

(A) Fluorescent microscopic images of XBP1s–GFP fusion constructs upon 24 h of transfection in HEK-293T cells. Nuclei were counter-stained with DAPI. wt, wild-type. (B) Immunoblots of XBP1 from immunoprecipitates (IP) with anti-FLAG–agarose prepared from HEK-293T cells transfected with Myc-tagged XBP1s with or without different FLAG–HDAC constructs (HDAC1–6). Note that same amount of plasmids were transfected in each condition. Inputs are shown in the lower two panels. (C) Immunoblots of XBP1 from immunoprecipitates (IP) with anti-FLAG–agarose prepared from HEK-293T cells transfected with FLAG–HDAC5 together with different HA–XBP1s constructs (left-hand panel) or Myc–XBP1s constructs (right-hand panel). Inputs for XBP1s and HDAC5 are shown (lower panels). s, XBP1; *, non-specific band. The molecular mass in kDa is indicated on the left-hand side.

SUMOylation does not affect the interaction between XBP1s and HDAC5

SUMOylation of transcription factors may also alter their interaction with transcriptional co-repressors such as HDACs [2729]. Indeed, out of all the HDACs (HDAC1–6), XBP1s strongly interacted with HDAC5 (Figure 5B). This interaction was not affected by mutations at the SUMOylation sites (Figure 5C). Thus our results show that XBP1s interacts with HDAC5 and this interaction is not affected by SUMOylation of XBP1s.

SUMOylation negatively regulates transcriptional activity of XBP1s

We next determined the physiological significance of SUMOylation on the transcriptional activity of XBP1s. First, we transfected wild-type or mutant XBP1s into XBP1−/− MEFs together with a luciferase reporter driven by a synthetic promoter containing four-tandem XBP1-binding sites (5′-TGACGT-3′) [30]. Wild-type XBP1s increased luciferase activity over 10-fold (Figure 6A). In line with the requirement of the C-terminal domain of XBP1s for transcriptional activation, loss of the C-terminal 100 amino acids of XBP1s (i.e. a truncated form of XBP1s protein) reduced the activity by 50% (Figure 6A). Mutations at either Lys276 or Lys297 increased XBP1s transcriptional activity by 50 and 100% relative to wild-type XBP1s respectively, whereas mutations at both residues further increased its transcriptional activity (Figure 6A). In contrast, mutation of Lys298 had no effect. Similar observations were obtained in HEK-293T cells (Figure 6A). It remains unclear why K276R/K297R XBP1s has higher transcriptional activity compared with K297R XBP1s as both mutants abolish SUMOylation of XBP1s (Figure 2B). We speculate that the Lys276 residue in the K297R mutant may still be SUMOylated at a low efficiency beyond the detection limit or be modified via a SUMO-independent mechanism, which may negatively regulates its transcriptional activity.

SUMOylation represses transcriptional activity of XBP1s

Figure 6
SUMOylation represses transcriptional activity of XBP1s

(A and B) Luciferase assay in transfected XBP1−/− MEF or HEK-293T cells transfected with various XBP1 constructs. Luciferase gene expression is driven by a synthetic promoter consisted of four tandem XBP1-binding sites. Truncated, XBP1s with no C-terminal transactivation domain. −, GFP control; wt, wild-type. (C) qRT-PCR analysis of UPR genes in XBP1−/− MEFs transfected with control GFP (−) or different XBP1s constructs. Results are means ± S.E.M. *, P<0.05 using an unpaired two-tailed Student's t-test comparing mutant XBP1s with wild-type XBP1s.

Figure 6
SUMOylation represses transcriptional activity of XBP1s

(A and B) Luciferase assay in transfected XBP1−/− MEF or HEK-293T cells transfected with various XBP1 constructs. Luciferase gene expression is driven by a synthetic promoter consisted of four tandem XBP1-binding sites. Truncated, XBP1s with no C-terminal transactivation domain. −, GFP control; wt, wild-type. (C) qRT-PCR analysis of UPR genes in XBP1−/− MEFs transfected with control GFP (−) or different XBP1s constructs. Results are means ± S.E.M. *, P<0.05 using an unpaired two-tailed Student's t-test comparing mutant XBP1s with wild-type XBP1s.

To exclude further the possibility of SUMO-independent effects at the two lysine residues (Lys276 and Lys297), we mutated the glutamate residue in the SUMO motifs (ΨKXE) and replaced it with an alanine or glutamine residue, i.e. E278A/Q and E299A/Q. These mutants exhibited similar transcriptional activity as the lysine mutant located in the same motif (Figure 6B), pointing to a key role of both SUMO motifs in regulating XBP1s activity. Thus these results demonstrate unequivocally that SUMOylation negatively regulates transcriptional activity of XBP1s.

We next determined the effect of SUMOylation on the expression of known XBP1s target genes. To this end, we transfected wild-type or mutant XBP1s into XBP1−/− MEFs and measured the expression of endogenous UPR genes. Mutations of Lys276 or Lys297, but not of Lys298, strongly induced the expression of several well-known XBP1 target genes [10], including Edem (ER degradation enhancer, mannosidase), Herp (homocysteine-induced endoplasmic reticulum protein) and P58ipk, by nearly 30–50%, but not the ATF6 target gene Grp78 (glucose-regulated protein 78) (Figure 6C). Taken together, our results show that SUMOylation of XBP1s down-regulates its transcriptional activity.

DISCUSSION

In the present study we show that a key transcription factor of the UPR signalling pathway is subjected to covalent post-translational modifications via SUMOylation. Our results showed that XBP1s protein, generated upon activation of IRE1α RNase activity during the ER stress response or UPR, is SUMOylated at two lysine residues in the C-terminal transactivation domain and, that these SUMOylation events have additive suppressive effects on XBP1s transcriptional activity. Thus the transcriptional activity of XBP1s protein is regulated not only by the IRE1α-mediated splicing event, but also by post-translational modifications, such as SUMOylation.

Our results adds XBP1s to a growing list of transcription factors whose activity is regulated by SUMOylation and suggests an unexpected role for SUMOylation in regulating ER homoeostasis and UPR activity. As SUMOylation is a highly transient post-translational modification event, it may serve as a mechanism to further fine-tune the transcriptional activity of XBP1s and, in turn, affect the outcome of the UPR. Nonetheless, similar to the scenario for most other transcription factors that are regulated by SUMOylation [19,20], the detailed molecular mechanism by which SUMOylation affects XBP1s transcriptional activity requires further investigation.

Previous studies have established the indispensible role of the IRE1α/XBP1 branch of the UPR in plasma cell and adipocyte differentiation [15,16,18,31]. During plasma cell differentiation, accumulation of XBP1s protein induces a series of transcriptional events leading to enlarged cell size, expanded ER membrane/volume and increased secretory capacity [32]. On the other hand, during adipocyte differentiation, accumulation of XBP1s protein increases the expression of C/EBPα, a key adipogenic factor, which subsequently controls cellular adaptation to a mature adipocyte phenotype [18]. Further studies are required to determine whether SUMOylation of XBP1s plays a role in cellular differentiation processes.

The UPR, a critical cellular defence mechanism against the accumulation of misfolded proteins and aggregates in the ER, has been implicated in many human diseases, collectively termed ‘conformational diseases’, including diabetes, cancer and neurodegeneration [33]. When the UPR is overwhelmed or fails, it often leads to cellular dysfunction and cell death [11,33]. As our knowledge of the role of UPR and XBP1s in human diseases improves [34,35], the ability to modulate the transcriptional activity of XBP1s may be an important first-step towards improving therapeutical strategies targeting dysfunctional UPR.

Abbreviations

     
  • ATF6

    activating transcription factor 6

  •  
  • ATF6N

    nuclear ATF6

  •  
  • C/EBPα

    CCAAT/enhancer-binding protein α

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DTT

    dithiothreitol

  •  
  • ER

    endoplasmic reticulum

  •  
  • GFP

    green fluorescent protein

  •  
  • Grp78

    glucose-regulated protein 78

  •  
  • HDAC

    histone deacetylase

  •  
  • HEK-293T

    HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)

  •  
  • HERP

    homocysteine-induced endoplasmic reticulum protein

  •  
  • HRP

    horseradish peroxidase

  •  
  • IRE1α

    inositol-requiring enzyme 1α

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • PEI

    polyethylenimiene

  •  
  • PIAS

    protein inhibitor of activated STAT

  •  
  • hPIAS

    human PIAS

  •  
  • mPIAS

    mouse PIAS

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • RanBP2

    Ran-binding protein 2

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • SUMO

    small ubiquitin-related modifier

  •  
  • Tg

    thapsigargin

  •  
  • UPR

    unfolded protein response

  •  
  • XBP1

    X-box-binding protein 1

  •  
  • XBP1s

    spliced XBP1

  •  
  • XBP1u

    unspliced XBP1

AUTHOR CONTRIBUTION

Hui Chen and Ling Qi conceived the project. Hui Chen performed the experiments and analysed the results. Ling Qi wrote the paper and with assistance from Hui Chen.

We thank Laurie Glimcher, Jorma Palvimo, Marc Montminy, Ling Cai and Katherine Kieckhafer for cell lines and plasmids; Yin He for critical reading of the manuscript; and other members of the Qi laboratory for insightful comments and suggestions.

FUNDING

This study was supported by the American Federation for Aging Research [grant number RAG08061] and the National Institute of Diabetes and Digestive and Kidney Disease [grant number R01DK082582]. L.Q. is the recipient of the Rosalinde and Arthur Foundation/American Federation for Aging Research New Investigator Award in Alzheimer's Diseases and the American Diabetes Association (ADA) Junior Faculty Award.

References

References
1
Mori
K.
Ma
W.
Gething
M. J.
Sambrook
J.
A transmembrane protein with a Cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus
Cell
1993
, vol. 
74
 (pg. 
743
-
756
)
2
Cox
J. S.
Shamu
C. E.
Walter
P.
Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase
Cell
1993
, vol. 
73
 (pg. 
1197
-
1206
)
3
Cox
J. S.
Walter
P.
A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response
Cell
1996
, vol. 
87
 (pg. 
391
-
404
)
4
Sidrauski
C.
Cox
J. S.
Walter
P.
tRNA ligase is required for regulated mRNA splicing in the unfolded protein response
Cell
1996
, vol. 
87
 (pg. 
405
-
413
)
5
Sidrauski
C.
Walter
P.
The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response
Cell
1997
, vol. 
90
 (pg. 
1031
-
1039
)
6
Calfon
M.
Zeng
H.
Urano
F.
Till
J. H.
Hubbard
S. R.
Harding
H. P.
Clark
S. G.
Ron
D.
IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA
Nature
2002
, vol. 
415
 (pg. 
92
-
96
)
7
Yoshida
H.
Matsui
T.
Yamamoto
A.
Okada
T.
Mori
K.
XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor
Cell
2001
, vol. 
107
 (pg. 
881
-
891
)
8
Lee
K.
Tirasophon
W.
Shen
X.
Michalak
M.
Prywes
R.
Okada
T.
Yoshida
H.
Mori
K.
Kaufman
R. J.
IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response
Genes Dev.
2002
, vol. 
16
 (pg. 
452
-
466
)
9
Shen
X.
Ellis
R. E.
Lee
K.
Liu
C. Y.
Yang
K.
Solomon
A.
Yoshida
H.
Morimoto
R.
Kurnit
D. M.
Mori
K.
Kaufman
R. J.
Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development
Cell
2001
, vol. 
107
 (pg. 
893
-
903
)
10
Lee
A. H.
Iwakoshi
N. N.
Glimcher
L. H.
XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response
Mol. Cell. Biol.
2003
, vol. 
23
 (pg. 
7448
-
7459
)
11
Ron
D.
Walter
P.
Signal integration in the endoplasmic reticulum unfolded protein response
Nat. Rev. Mol. Cell Biol.
2007
, vol. 
8
 (pg. 
519
-
529
)
12
Lee
A. H.
Chu
G. C.
Iwakoshi
N. N.
Glimcher
L. H.
XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands
EMBO J.
2005
, vol. 
24
 (pg. 
4368
-
4380
)
13
Masaki
T.
Yoshida
M.
Noguchi
S.
Targeted disruption of CRE-binding factor TREB5 gene leads to cellular necrosis in cardiac myocytes at the embryonic stage
Biochem. Biophys. Res. Commun.
1999
, vol. 
261
 (pg. 
350
-
356
)
14
Reimold
A. M.
Etkin
A.
Clauss
I.
Perkins
A.
Friend
D. S.
Zhang
J.
Horton
H. F.
Scott
A.
Orkin
S. H.
Byrne
M. C.
, et al. 
An essential role in liver development for transcription factor XBP-1
Genes Dev.
2000
, vol. 
14
 (pg. 
152
-
157
)
15
Reimold
A. M.
Iwakoshi
N. N.
Manis
J.
Vallabhajosyula
P.
Szomolanyi-Tsuda
E.
Gravallese
E. M.
Friend
D.
Grusby
M. J.
Alt
F.
Glimcher
L. H.
Plasma cell differentiation requires the transcription factor XBP-1
Nature
2001
, vol. 
412
 (pg. 
300
-
307
)
16
Zhang
K.
Wong
H. N.
Song
B.
Miller
C. N.
Scheuner
D.
Kaufman
R. J.
The unfolded protein response sensor IRE1α is required at two distinct steps in B cell lymphopoiesis
J. Clin. Invest.
2005
, vol. 
115
 (pg. 
268
-
281
)
17
Lee
A. H.
Scapa
E. F.
Cohen
D. E.
Glimcher
L. H.
Regulation of hepatic lipogenesis by the transcription factor XBP1
Science
2008
, vol. 
320
 (pg. 
1492
-
1496
)
18
Sha
H.
He
Y.
Chen
H.
Wang
C.
Zenno
A.
Shi
H.
Yang
X.
Zhang
X.
Qi
L.
The IRE1α–XBP1 pathway of the unfolded protein response is required for adipogenesis
Cell Metab.
2009
, vol. 
9
 (pg. 
556
-
564
)
19
Hay
R. T.
SUMO: a history of modification
Mol. Cell
2005
, vol. 
18
 (pg. 
1
-
12
)
20
Rytinki
M. M.
Kaikkonen
S.
Pehkonen
P.
Jaaskelainen
T.
Palvimo
J. J.
PIAS proteins: pleiotropic interactors associated with SUMO
Cell. Mol. Life Sci.
2009
, vol. 
66
 (pg. 
3029
-
3041
)
21
Golebiowski
F.
Matic
I.
Tatham
M. H.
Cole
C.
Yin
Y.
Nakamura
A.
Cox
J.
Barton
G. J.
Mann
M.
Hay
R. T.
System-wide changes to SUMO modifications in response to heat shock
Sci. Signaling
2009
, vol. 
2
 pg. 
ra24
 
22
Johnson
E. S.
Protein modification by SUMO
Annu. Rev. Biochem.
2004
, vol. 
73
 (pg. 
355
-
382
)
23
Oishi
Y.
Manabe
I.
Tobe
K.
Ohsugi
M.
Kubota
T.
Fujiu
K.
Maemura
K.
Kubota
N.
Kadowaki
T.
Nagai
R.
SUMOylation of Kruppel-like transcription factor 5 acts as a molecular switch in transcriptional programs of lipid metabolism involving PPAR-δ
Nat. Med.
2008
, vol. 
14
 (pg. 
656
-
666
)
24
Stankovic-Valentin
N.
Deltour
S.
Seeler
J.
Pinte
S.
Vergoten
G.
Guerardel
C.
Dejean
A.
Leprince
D.
An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity
Mol. Cell. Biol.
2007
, vol. 
27
 (pg. 
2661
-
2675
)
25
Duprez
E.
Saurin
A. J.
Desterro
J. M.
Lallemand-Breitenbach
V.
Howe
K.
Boddy
M. N.
Solomon
E.
de The
H.
Hay
R. T.
Freemont
P. S.
SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation
J. Cell Sci.
1999
, vol. 
112
 (pg. 
381
-
393
)
26
Rodriguez
M. S.
Dargemont
C.
Hay
R. T.
SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
12654
-
12659
)
27
Girdwood
D.
Bumpass
D.
Vaughan
O. A.
Thain
A.
Anderson
L. A.
Snowden
A. W.
Garcia-Wilson
E.
Perkins
N. D.
Hay
R. T.
P300 transcriptional repression is mediated by SUMO modification
Mol. Cell
2003
, vol. 
11
 (pg. 
1043
-
1054
)
28
Shiio
Y.
Eisenman
R. N.
Histone sumoylation is associated with transcriptional repression
Proc. Natl. Acad. Sci. U.S.A.
2003
, vol. 
100
 (pg. 
13225
-
13230
)
29
Yang
S. H.
Sharrocks
A. D.
SUMO promotes HDAC-mediated transcriptional repression
Mol. Cell
2004
, vol. 
13
 (pg. 
611
-
617
)
30
Clauss
I. M.
Chu
M.
Zhao
J. L.
Glimcher
L. H.
The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core
Nucleic Acids Res.
1996
, vol. 
24
 (pg. 
1855
-
1864
)
31
Iwakoshi
N. N.
Lee
A. H.
Vallabhajosyula
P.
Otipoby
K. L.
Rajewsky
K.
Glimcher
L. H.
Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1
Nat. Immunol.
2003
, vol. 
4
 (pg. 
321
-
329
)
32
Shaffer
A. L.
Shapiro-Shelef
M.
Iwakoshi
N. N.
Lee
A. H.
Qian
S. B.
Zhao
H.
Yu
X.
Yang
L.
Tan
B. K.
Rosenwald
A. et al.
XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation
Immunity
2004
, vol. 
21
 (pg. 
81
-
93
)
33
Yoshida
H.
ER stress and diseases
FEBS J.
2007
, vol. 
274
 (pg. 
630
-
658
)
34
Kaser
A.
Lee
A. H.
Franke
A.
Glickman
J. N.
Zeissig
S.
Tilg
H.
Nieuwenhuis
E. E.
Higgins
D. E.
Schreiber
S.
Glimcher
L. H.
Blumberg
R. S.
XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease
Cell
2008
, vol. 
134
 (pg. 
743
-
756
)
35
Kim
I.
Xu
W.
Reed
J. C.
Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities
Nat. Rev. Drug Discov.
2008
, vol. 
7
 (pg. 
1013
-
1030
)

Supplementary data