SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes¹

Activator protein-1 collectively describes a class of structurally and functionally related proteins that are characterized by a basic DNA-binding domain and a leucine-zipper dimerization motif. It principally comprises members of the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) protein families. These proteins bind as dimers to specific sequences in the promoter and enhancer regions of target genes; the canonical site mediates activation by 12-O-tetradecanoylphorbol-13-acetate (TPA)⁵ and is termed a TPA response element (TRE). Additionally, some members of the activating transcription factor and Jun dimerization protein families can form AP-1 complexes, primarily with Jun proteins, and bind to TRE-like sequences (1, 2). Unlike the Jun proteins, Fos family members cannot form homodimers, but heterodimerize with Jun partners, giving rise to transactivating or transrepressing complexes with different biochemical properties.

Jun proteins are implicated in numerous cellular processes, such as transcriptional control, proliferation, differentiation, and tumorigenesis (3, 4, 5, 6). In particular, JunB has a unique, nonredundant function in vivo, because its inactivation in mice causes vascular defects in extra-embryonic tissues that lead to embryonic lethality (7). In adult mice, JunB plays an important regulatory role in the hematopoietic system and tumorigenesis. The embryonic lethality of JunB^−/− mice is reversed by expression of a junB transgene under the control of the Ubiquitin promoter (Ubi-JunB). However, these animals develop a myeloid hyperproliferation that progresses to a disease resembling chronic myeloid leukemia; remarkably, this corresponds to silencing of the Ubi-JunB transgene (8, 9). In agreement with this, junB expression is diminished in some human chronic myeloid leukemia (10) and B cell leukemias (11, 12). Similarly, JunB expression blocks leukemia induced by knockdown of PU.1 in mouse hematopoietic stem cells (13). Although this suggests that JunB is a tumor suppressor, it appears to be oncogenic in T cells, because some hyperproliferative T cell lymphomas show JunB overexpression (14, 15, 16, 17, 18). Accordingly, JunB is a critical regulator of the expression of cytokines important for T lymphocyte proliferation and differentiation, namely IL-4 (5, 19, 20) and IL-2 (21, 22, 23). Moreover, JunB expression and binding capacity are decreased in anergic T cells, which do not produce IL-2 (24, 25, 26, 27).

The activity of AP-1 is critically modulated by post-translational modifications, in particular, phosphorylation mediated by MAPK cascades. The JNK pathway is an important regulator of c-Jun function; however, its role in JunB phosphorylation remains unclear, even though JunB contains a docking site for JNKs (5, 28, 29). Instead, JNKs regulate the E3 ubiquitin ligase Itch that targets JunB for ubiquitylation in T cells (30). Thus, mice lacking Itch show an accumulation of JunB in helper T cells, which leads to an increase in Th2 differentiation (31).

Sumoylation is a reversible modification that conjugates an ubiquitin-like peptide, SUMO (for small ubiquitin-like modifier), onto specific lysine residues of substrate proteins (32, 33, 34). Three SUMO isoforms are expressed in mammalian cells. SUMO-1 shows 47% homology at the protein level with SUMO-2 and -3, whereas SUMO-2 and -3 are 95% homologous (33). Sumoylation of target proteins is a multistep process that is mechanistically analogous to ubiquitination. An E1-activating enzyme, namely Aos1/Uba2, and an E2-conjugating enzyme, Ubc9, are sufficient for SUMO conjugation in vitro. In vivo, this sometimes requires E3 factors, such as PIAS proteins, PC2 and RanBP2 (32). Sumoylation affects target protein function in a variety of ways and has now been implicated in many cellular processes, notably regulation of gene expression (33, 35). Numerous proteins involved in transcriptional control are sumoylated, including transcription factors, coactivators, corepressors, and histones, to name a few. In most cases, sumoylation represses transcription; the mechanisms include recruiting repressor complexes directly on promoters (35) or by sequestering proteins in nuclear subcompartments. Although sumoylation represses the activity of the AP-1 components c-Fos and c-Jun (36), it enhances that of other transcription factors, such as NF-AT1 and Oct4 (37, 38).

In the present study, we show that endogenous and ectopically expressed JunB is sumoylated in both resting and activated T lymphocytes. Of the three SUMO consensus sites, the site containing lysine 237 is the primary site of conjugation of either SUMO-1 or -2 in T cells. Remarkably, blocking JunB sumoylation, either by mutation or by coexpression with inactive Ubc9, strongly diminishes JunB-mediated transactivation of reporter genes controlled by the IL-2 and IL-4 promoters. In contrast, JunB sumoylation plays no role in its activity on synthetic TRE or AP-1 binding sites derived from the IL-2 promoter. These data suggest that sumoylation of JunB plays an important role in the transcriptional activation of certain cytokine genes in T cells.

Human hemagglutinin (HA)-tagged JunB wild type (wt), the single mutants (K237R, K267R, and K301R) and the JunB triple mutant (K237R/K267R/K301R, termed JunB-3R) were cloned in the pcDNA3 vector (Invitrogen). Point mutations were made using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The expression vectors pcDNA3-His₆-SUMO-2 and pCDNA3-His₆-SUMO-1 (39), the TRE-luciferase reporter (40), and IL-2 Luc reporter containing the 300bp upstream of the initiation site (41) have been described. The IL-4 Luc reporter plasmid was provided by Dr. Glimcher (42). wt Ubc9 in pCDNA3 was previously described (36). Dominant negative Ubc9 (Ubc9 DN) was obtained by mutating cysteine 93 to serine and leucine 97 to serine.

The anti-human CD3, anti-mouse CD3, and anti-mouse CD28 mAbs were purified as previously described (43, 44). The anti-human CD28 mAb was from BD Biosciences. The JunB rabbit polyclonal Ab was from Santa Cruz Biotechnology, and the mouse monoclonal JunB Ab was a kind gift from the laboratory of Dr. M. Yaniv. The rat monoclonal anti-HA (3F10) was from Roche. Donkey anti-rabbit or sheep anti-mouse IgG Abs were from Amersham Biosciences, and the goat anti-hamster IgG was from Pierce. N-ethylmaleimide (NEM), iodoacetamide, imidazole, PMA, DTT, and the ionomycin were from Sigma-Aldrich. The “Complete” protease inhibitor mixture was from Roche.

All cells were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES [pH 7.4], 1× MEM nonessential amino acid (Invitrogen), 50 μM 2-ME, and 100 U/ml each of penicillin G and streptomycin. Jurkat cells in logarithmic growth phase were transfected with the indicated amounts of plasmid by electroporation (40). In each experiment, cells were transfected with the same total amount of DNA by adding the required quantities of empty vector. Cells were incubated for 10 min at room temperature with the DNA mix and electroporated at 260 mV, 960 mF in 400 μl of RPMI 1640. Murine primary T cells were isolated from spleens using a murine T cell negative isolation kit (Dynal Biotech) according to the manufacturer’s protocol. Human PBMCs were isolated as previously described (45). Human CD4⁺ T cells were isolated from fresh whole blood using RosetteSep human CD4⁺ T cell enrichment (Stem Cell Technologies) according to the supplier’s protocol. Five to ten million CD4⁺ T cells were transfected using a Human T cell Nucleofector kit (Amaxa) following the manufacturer’s protocol. When required, CD4⁺ T cells were activated 6 h post-transfection as described below.

Plates were coated with 2 μg/ml each anti-human or -mouse CD3 and CD28 Abs in PBS for 4 h at 4°C. T cells were incubated on these plates for the indicated times. Plates were placed on ice, and cells were lysed with SDS-PAGE sample buffer (2% SDS, 0.1 M DTT, 50 mM Tris [pH 6.8], and 10% glycerol). PMA (100 ng/ml) and ionomycin (1 μg/ml) were added to the cell suspension for the indicated times. Cells were washed once with PBS and processed.

In all experiments, cells were transfected with a β-galactosidase reporter plasmid as control of transfection (40). Transfected cells (1 × 10⁶) were harvested after 2 days and washed twice with PBS. Cells were lysed in 100 μl luciferase lysis buffer (Promega) and luciferase assays (40 μl) performed according to the manufacturer’s instructions (Promega) using a Berthold luminometer. For β-galactosidase assays, 40 μl of lysates were added to 200 μl of β-galactosidase assay buffer (50 mM phosphate buffer [pH 7.4], ortho-nitro-phenyl-galactopyranoside 200 μg, 1 mM MgCl₂, and 50 mM 2-ME) and the absorbance measured at 400 nm. The results were expressed as luciferase units normalized to the corresponding β-galactosidase activity. The expression level of the transfected proteins was routinely controlled by immunobloting analysis.

Small-scale biochemical fractionation was performed as described previously (46). Briefly, 20 × 10⁶ cells were collected, washed with precold PBS, and resuspended in 0.5 ml of buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, “complete” protease inhibitor mixture, and 20 mM NEM). After addition of Triton X-100 (0.1% final concentration), cells were incubated on ice for 8 min, and nuclei (fraction P1) were collected by centrifugation (5 min, 1300g, 4°C). The supernatant (fraction S1) was clarified by high-speed centrifugation (5 min, 20000g, 4°C), and the supernatant (fraction S2) was collected. The P1 nuclei were washed once in buffer A and lysed on ice for 30 min in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitor mixture, 20 mM NEM, and 20 mM iodoacetamide). The insoluble chromatin (fraction P3) and soluble (fraction S3) fractions were separated by centrifugation (5 min, 1.700g, 4°C). The P3 fraction was washed once with buffer B and resuspended in SDS-PAGE sample buffer.

Forty million cells were lysed in 2 ml of Solution A (6 M guanidium-HCl, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.1 M Tris-HCl [pH 8.0], 20 mM NEM, 20 mM iodoacetamide, and 10 mM imidazole). Samples were sonicated (30 s) and centrifuged for 15 min at 14,000 rpm at 4°C. Supernatants were transferred to a new tube containing 30 μl of prewashed Ni²⁺-NTA magnetic beads (Promega) and incubated for 4 h at room temperature on a rocking platform. Twenty five μl of the supernatants were used as inputs. Beads were sequentially washed with 2 ml of the following solutions: A, B (8 M urea, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.1 M Tris-HCl [pH 8.0], 20 mM NEM, 20 mM iodoacetamide, and 10 mM imidazole), and C (8 M urea, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.1 M Tris-HCl [pH 6.3], 20 mM NEM, 20 mM iodoacetamide, 10 mM imidazole, and 0.2% Triton X-100). After two additional washes in buffer C without Triton X-100, proteins were eluted by adding 50 μl of SDS-PAGE sample buffer and analyzed by SDS-PAGE.

Total RNA was isolated from 3 × 10⁶ human CD4⁺ T cells using a GenElute Mammalian total RNA Miniprep kit (Sigma-Aldrich) using the supplier’s instructions. One to 2 μg of RNA were treated with DNase I (Promega), and 1 μg RNA was used to synthesize cDNA using pdN9 primers and M-Mulv reverse transcriptase (New England Biolabs) using the supplier’s protocol. Q-PCR for IL-2 and S26 was performed using Taq polymerase and SYBR green reagent (Invitrogen) according to the manufacturer’s instructions. The Q-PCR was performed in a 96-well plate using the Stratagene MX3000P, and analysis was performed with MXPro software. All samples were analyzed in duplicates, and the results were expressed as fold induction compare with the empty pCDNA₃ vector. Primers used in this study were hIL-2, sense: TCA CCA GGA TGC TCA CAT TTA AGT; antisense: GA GGT TTG AGT TCT TCT TCT AGA CAC TGA; and for hRPS26, sense: GAA CGC ATT TCC ACC CTA GA; antisense: GCA CGA CCA TTG TTC CTT CT.

Human CD4⁺ T cells were seeded at 2.10⁶ cells/ml in 24-well plates (Pharmingen) precoated with 2 μg/ml anti-CD3 and anti-CD28 Abs and left at 37°C for 12 h. IL-2 secretion was measured using Human IL-2 quantikine ELISA kit (R&D systems) following the manufacturer’s instructions. All samples were analyzed in duplicates, and the results were expressed as fold induction compare with cells transfected with empty pcDNA₃ vector.

The statistical analysis of the differences between means of paired samples was performed using the paired t test. The results are given as the confidence interval (p). All the experiments described in the figures were performed at least three times with similar results.

JunB migrates in SDS-PAGE as a doublet with an apparent molecular mass of 40 kDa in the murine leukemic T cell line EL4, as well as in mouse and human primary T cells (Fig. 1). This complements the list of resting T cells of different origins that express this protein constitutively (5, 25, 27). In EL4 cells, activation with PMA/ionomycin treatment increases JunB expression, as previously described in other cells (47, 48). Moreover, we observed a species of JunB migrating at 60 kDa that is more easily visualized after PMA/ionomycin treatment (Fig. 1,A). This band was also observed when we used a mouse monoclonal JunB Ab against the JunB N-terminal. The 20 kDa increase in size indicated a post-translational modification, such as sumoylation, which has already been described for ectopically expressed JunB in Hela cells (49). Importantly, the 60 kDa JunB band was also observed in mouse and human primary T cells activated either with PMA/ionomycin or anti-CD3/anti-CD28. Maximal activation with the latter was observed 2 h after stimulation; at 1 h, we found a small increase in nonmodified and modified proteins (Fig. 1 B).

To confirm that this band comigrates with sumoylated JunB, we cotransfected Jurkat T cells with expression vectors for His₆-SUMO-2 and the SUMO E2 enzyme Ubc9. After activation with PMA/Ionomycin, cells were lysed under denaturing conditions to prevent desumoylation, and SUMO-2 conjugates were purified by metal affinity chromatography. A fraction of endogenous JunB was detected in the latter by immunoblot analysis, where it migrated at 60 kDa (Fig. 1,C). This strongly suggests that the 60 kDa band observed in Fig. 1, A and B, represents monosumoylated JunB.

Most studies have needed to use protein enrichment or overexpression of the protein or SUMO isoforms to detect the sumoylated forms. These include the transcription factors NF-AT, Oct-4, and Elk-1 (38, 50, 51). In contrast, we readily detected the upper JunB band in total cell extracts without enrichment of sumoylated proteins or ectopic expression of JunB or SUMO isoforms. Hence, the proportion of endogenous JunB appearing to be sumoylated is higher than many other proteins.

To facilitate further molecular analysis, we checked whether ectopically expressed JunB is also sumoylated in Jurkat cells. Transfections were performed as described above with the addition of an expression vector for HA-tagged JunB wt. Affinity-purified His-tagged proteins were separated by SDS-PAGE, and ectopic JunB was visualized in Western blots by immunodetection of the HA epitope. Like the endogenous protein, a proportion of exogenous JunB was conjugated to SUMO-2 in both resting and activated T cells (Fig. 2,A). Similar results were obtained using an expression vector for His₆-SUMO-1 (Fig. 3,B). Although JunB was predominantly monosumoylated, SUMO-2 also generated a ladder reflecting conjugation of multiple peptides (Fig. 2 A, upper panel). This could arise from addition of SUMO-2 chains to a single site in JunB, monosumoylation on multiple sites, or a combination of the two (see below).

Sumoylation generally occurs on the lysine residue within a ψKXE consensus motif, where ψ is an aliphatic amino acid and X any amino acid (33). Three motifs are present in JunB, surrounding lysines 237, 267, and 301 (Fig. 2,B). To determine which sites are used in vivo, these lysines were mutated to arginines individually (JunB-K237R, -K267R, and -K301R, Fig. 2,B) or in combination (JunB-K237R/K267R/K301R, termed JunB-3R). The expression vectors were transfected into Jurkat cells, which were then activated by PMA/ionomycin and lysed. Overexpressed wt or mutant JunB in the whole cell extracts was detected in Western blots using an anti-HA Ab. Mutation of lysine 237, but not that of lysines 267 or 301, strongly diminished JunB modification by endogenous SUMO (Fig. 3,A). Nevertheless, these two lysines were used, albeit weakly, because the residual level of the 60 kDa band seen with JunB-K237R completely disappeared in JunB-3R (Fig. 3,A). To confirm this, we overexpressed either JunB wt, K237R, or 3R in Jurkat cells together with His₆-SUMO-1 or His₆-SUMO-2 and purified SUMO conjugates from cell lysates by nickel affinity chromatography under denaturing conditions (Figs. 2,A and 3,B). Indeed, mutation of lysine 237 strongly impaired JunB conjugation with SUMO-1 (Fig. 3,B) or SUMO-2 (data not shown). A low level of sumoylation was still found with JunB-3R (Fig. 2 A), indicating the presence of another weak, nonconsensus site in JunB. Taken together, these data show that lysine 237 in JunB is the preferred conjugation site for SUMO.

SUMO conjugation has been linked to nuclear localization of numerous transcription factors (33), such as the T cell regulatory protein NF-AT1 (38). Moreover, certain sumoylated transcriptional regulators are found associated with a nuclear subdomain that is implicated in leukemia, namely promyelocytic leukemia nuclear bodies (Ref. 33). Indirect immunofluorescence microscopy showed endogenous JunB to be primarily nuclear in primary T cells and Jurkat cells, without revealing any preferential subnuclear association (data not shown). This confirms fractionation experiments in Jurkat cells, where JunB was exclusively in the nucleus (40).

Like JunB, the AP-1 family members c-Jun and c-Fos are conjugated to SUMO (36, 49), and sumoylated c-Fos preferentially localizes in an insoluble nuclear fraction in HeLa cells (36). To investigate whether this was also the case for sumoylated JunB, we performed nuclear fractionation experiments (Fig. 4). Because we wished to localize sumoylated vs nonsumoylated endogenous JunB in activated vs nonactivated cells, we performed a 1-h stimulation of PBMCs with anti-CD3/CD28 Abs (Fig. 1). Although unconjugated JunB was equally distributed in the soluble and insoluble nuclear fractions (S2 and P3, respectively, left panels), sumoylated JunB was detectable uniquely in the insoluble fraction. The purity of the two fractions was confirmed by the selective presence of the chromatin- and matrix-associated protein Topoisomerase I in P3 and the nucleoplasmic protein PHAX in S2. This indicates that SUMO-conjugated JunB associates with the insoluble fraction of the nucleus, possibly a subdomain tightly associated with chromatin. In addition, we detected similar levels of the 60 kDa band representing endogenous JunB conjugated to SUMO (Fig. 4, right panel) in resting and stimulated cells. These data show that T cell activation did not increase the percentage of sumoylated JunB or change JunB intracellular localization. The sumoylated form of JunB was easily observed after activation because activated cells expressed more JunB (Fig. 1). Consistent with this, ectopically expressed JunB was sumoylated to a similar extent in resting Jurkat cells as endogenous JunB in activated cells (Fig. 3). Therefore, sumoylation of JunB was constitutive and did not require T cell activation; still, as the total amount of JunB increased in activated T cells via the de novo expression of the protein (Fig. 1), the amount of sumoylated JunB increased comparably.

Sumoylation regulates transactivation by numerous transcription factors and, in many cases, has a repressive effect (35). To evaluate the effect of SUMO conjugation on trans-activation by JunB, we performed cotransfection experiments with reporter genes controlled by the IL-2 and IL-4 promoters, two well-known JunB targets in T cells (5, 22, 52). In Jurkat cells, wt JunB activated the IL-2 promoter by 70-fold (Fig. 5,A). Interestingly, induction was half as strong with JunB-K237R, and mutation of all three consensus lysines almost abolished activation of the IL-2 promoter. All three proteins were expressed at similar levels (Fig. 5,A). Our results show that K237 was the primary site for sumoylation; when this site was mutated to alanine, we detected a minor level of sumoylated JunB (Fig. 3). This indicates that other lysines, especially K267 and K301 in the other sumoylation consensus sites, were also conjugated with SUMO. Therefore, we used the triple mutant that was barely sumoylated to investigate the role of sumoylation on the transcriptional activity of JunB. This mutant activated the TRE, NF-AT/AP-1, and CD28 reporters to the same extent as wt JunB, indicating that the mutation did not affect basal function (Fig. 6). In agreement with Li et al. (5), wt JunB activated the IL-4 reporter gene 4-fold (Fig. 5,C); this was reduced to control levels with JunB-3R (Fig. 5 C). These data suggested that sumoylation of JunB potentiates its ability to transactivate the IL-4 and IL-2 promoters.

Ubc9 is the only SUMO-E2 identified in human cells. As expected, cotransfection of Ubc-9DN led to inhibition of sumoylation of ectopically expressed JunB (Fig. 5,B). Consistent with the results using nonsumoylable mutants, cotransfection of Ubc-9DN led to a 2-fold reduction in JunB-driven activation of the IL-2 reporter gene (Fig. 5 B). Although this strongly suggests that Ubc9-DN inhibits JunB activity in this context, we cannot exclude that part of the Ubc9-DN effect was mediated by an effect on another protein.

Surprisingly, wt JunB and JunB-3R showed indistinguishable activity on the reference AP-1 reporter gene, namely the TRE reporter plasmid that is driven by four canonical binding sites upstream of the TATA box (Fig. 6,A). Thus, sumoylation regulates JunB activity on specific cytokine promoters. The proximal region of the IL-2 promoter contains several regulatory elements targeted by different transcription factors (23). The composite elements ARRE-2 and CD28RE are recognized by NF-AT and NF-κB, respectively, in combination with AP-1, in particular JunB (22, 25, 26). Surprisingly, JunB-K237R and JunB-3R transactivated reporter genes driven by these composite sites at the same level as wt JunB (Fig. 6 B and C). Taken together, these results suggest that sumoylation controls JunB transcriptional activity on certain cytokine promoters without intrinsically affecting JunB transactivation.

To unambiguously investigate the role of JunB sumoylation in primary T cells, we transfected human CD4⁺ T cells with JunB and JunB-3R. Resting CD4⁺ T cells did not express measurable levels of IL-2, and ectopically expressed JunB did not induce its expression (data not shown). Therefore, in the next set of experiments, we used activated CD4⁺ T cells. Like in Jurkat T cells, ectopically expressed JunB wt, but not JunB-3R, was sumoylated in CD4⁺ T cells (Fig. 7,A). wt JunB, but not JunB-3R, significantly increased expression of endogenous IL-2 mRNA in CD4⁺ T cells (Fig. 7,B). We confirmed this differential effect of JunB wt and -3R by measuring IL-2 levels in supernatants of transfected cells (Fig. 7 C). Thus, in primary CD4⁺ T cells, sumoylation of JunB cooperates with other endogenous signals to fully activate the IL-2 promoter.

We report here that JunB is covalently modified by either SUMO-1 or SUMO-2 in both human and mouse T cells. The major acceptor site is lysine 237, although weak levels of sumoylation are detectable on other sites. Like the majority of sumoylated proteins (32, 33, 35), a minor proportion of JunB, both endogenous and exogenous, was conjugated with SUMO under steady-state conditions. However, we could detect sumoylated JunB in whole cell extracts, which was enhanced by enrichment of SUMO-conjugated proteins. Activation of T cells also allowed us to more easily visualize sumoylated JunB. This reflected the increase in JunB expression, because the ratio between sumoylated and nonsumoylated JunB remained the same as found in resting cells. The latter observation suggests that this modification of JunB is constitutive and does not require T cell activation. However, this does not exclude that other signals or stresses could regulate JunB sumoylation, as observed for c-Fos and c-Jun in Hela cells (36, 39).

Sumoylation is often linked to a specific intranuclear distribution of the target proteins. Many sumoylated nuclear proteins localize in nuclear subdomains, such as promyelocytic leukemia nuclear bodies, which may serve to sequester them and thereby suppress their activity. Endogenous JunB is nuclear in primary T cells and Jurkat cells but showed no concentration in a subdomain. Nevertheless, the modified protein does show a striking localization, as it was found exclusively in the insoluble nuclear fraction of human primary T cells, i.e., associated with a detergent-resistant nuclear matrix and/or chromatin structure. In contrast, nonmodified JunB was equally distributed between soluble and nonsoluble nuclear chromatin fractions. Thus, JunB resembles c-Fos and several other nuclear factors in the preferential binding of the sumoylated protein to this insoluble nuclear structure (36, 53). Further work will determine whether sumoylation is a cause or a consequence of this localization.

In certain transcriptional regulators, such as Elk-1, the lysines in the sumoylation consensus motif were initially identified as being essential for repressing their activity (54). Similarly, this motif is found in synergy control domains present in c-Myb, C/EBP, SP3, and nuclear receptors (55). Synergy controls down-regulate synergistic activation of reporter genes driven by compound transcriptional regulatory elements but do not affect the activity of single response elements. Subsequently, this negative regulation was shown to be dependent upon sumoylation, and, in fact, SUMO conjugation to most transcription factors, including c-Fos and c-Jun, represses their activity (36, 49). It is striking that we observe the opposite result with JunB, whose ability to transactivate reporter genes driven by cytokine promoters was strongly compromised by blocking sumoylation, either by mutation or using DN-Ubc9. Thus, JunB joins the small subset of transcription factors, such as Oct4 (37), NF-AT (38), and NF-E2 (56), whose activity is potentiated by SUMO conjugation. JunB appears unique in that sumoylation is not a prerequisite for its transcriptional activity per se, because the mutants JunB-K237R and JunB-3R resemble JunB wt in the activation of reporter genes driven by isolated AP-1 binding sites of the IL-2 promoter.

How might sumoylation affect JunB transcriptional activity on cytokine promoters but not isolated elements? It could be that sumoylated JunB is targeting a different regulatory element than AP-1, NF-AT, or NF-κB, or that sumoylated JunB interacts with different proteins than nonsumoylated JunB. This will be difficult to establish, because JunB might interact with several AP-1 components, e.g., c-Jun and c-Fos. Moreover, the resulting AP-1 dimer might interact with other transcription factors, e.g., NF-AT or NF-κB, as well as coactivators. Given the multiple AP-1 sites in the IL-2 and IL-4 promoters, we cannot rule out that sumoylation of JunB subtly regulates its binding to different sites. Nevertheless, there are other potential explanations. For example, JunB sumoylation might facilitate recruitment and assembly of an enhanceosome on the promoter that is also dependent upon other transcriptional regulators, clearly present under our experimental conditions. There is some precedent for protein-protein interactions dependent upon SUMO conjugation (55). It is also possible that sumoylated JunB directs its target gene to a nuclear subcompartment of highly active transcription, or transcription factory (57); this might be reflected by the presence of SUMO-conjugated JunB in the detergent-insoluble fraction of the nucleus. Once again, strong transcriptional activation would then require promoter assembly with other transcriptional regulators induced by T cell activation. Any of these could explain the strong activation of the IL-2 promoter by sumoylated JunB.

In the immune system, this would be particularly important in T cell anergy, where a defect in JunB-dependent transcription leads to impaired IL-2 production (24). We would argue that a failure to sumoylate JunB would have the same consequence, namely a weak activation of IL-2 resulting in anergy. This is supported by our findings that nonsumoylable JunB mutants fail to increase IL-2 production in activated CD4⁺ T cells. This means that JunB needs to be sumoylated to fully activate the IL-2 promoter. We note that ectopically expressed JunB does not activate the IL-2 promoter in resting cells; this indicates that other factors or signals are required. Further work will be required to prove this hypothesis and characterize the mechanism by which SUMO enhances JunB activity on cytokine promoters.

We thank the members of the Intracellular Signaling and Gene Regulation group, along with Dr. Isabelle Jariel-Encontre, for helpful discussions and comments during this work. We also thank Dr. C. Duperray for help on cell sorting and Dr. Monique Dao for help in human CD4⁺ T cell isolation and transfection. We gratefully acknowledge the invaluable expertise of the members of the Animal Facility at the Institut de Génétique Moléculaire de Montpellier.

The authors have no financial conflict of interest.