PIASy, a nuclear matrix–associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies

PIASy-associated LEF1 is modified by SUMO conjugation

The appearance of an 18-kD-larger, slower-migrating form of LEF1 in cells expressing both LEF1 and PIASy raised the question as to whether the coexpression of these proteins results in the covalent modification of LEF1 by conjugation with ubiquitin or with the small ubiquitin-like modifier SUMO. Toward this end, we probed three parallel blots of T7-PIASy–coimmunoprecipitated proteins with anti-ubiquitin, anti-SUMO1, or anti-SUMO2/3 antiserum and subsequently reprobed these blots with anti-LEF1 antiserum. Coimmunoprecipitated LEF1 protein did not react with the anti-ubiquitin antiserum or with the anti-SUMO1 antibody, but the slower-migrating form of LEF1 reacted with the anti-SUMO2/3 antiserum (Fig. ​(Fig.2A;2A; data not shown). The anti-SUMO2/3 immunoblot also showed that many SUMO-modified proteins are coimmunoprecipitated from lysates of cells transfected with the T7-PIASy expression construct. In parallel immunoprecipitation experiments under more stringent conditions, we were unable to detect any SUMO2/3-modified proteins, suggesting that these proteins represent PIASy-associated proteins rather than SUMO-modified forms of PIASy (data not shown). Immunoblot analysis of total cell lysates with anti-LEF1 antiserum or anti-T7 antibody showed similar expression levels of LEF1 and PIASy in the transfected cells (Fig. ​(Fig.2A,2A, lower panels).

Open in a separate window

Figure 2

LEF1 is SUMO-modified in vivo. (A) PIASy associates with SUMO-modified LEF1. LEF1 and T7-PIASy were transiently expressed in 293T cells. Equivalent amounts of total cellular protein were immunoprecipitated with anti-T7 mAb. Coimmunoprecipitated endogenous, SUMO-conjugated proteins were detected by an anti-SUMO2/3 immunoblot (upper left). The anti-SUMO2/3 immunoblot was stripped and reprobed with an anti-LEF1 antibody (upper right). Numbers to the right indicate the molecular mass of protein markers in kilodaltons. The expression of LEF1 or T7-PIASy proteins in total cell lysates was determined by anti-LEF1 (middle) or anti-T7 (bottom) immunoblots, respectively. (B) LEF1 is SUMO-modified in vivo. LEF1, T7-PIASy, and Flag epitope-tagged SUMO1 or SUMO2 were transiently expressed in 293T cells. Equivalent amounts of total cellular protein were immunoprecipitated with anti-T7 mAb. Coimmunoprecipitated LEF1 proteins were detected by an anti-LEF1 immunoblot (top panel). The expression of LEF1 or T7-PIASy proteins in total cell lysates was determined by anti-LEF1 (middle) or anti-T7 (bottom) immunoblots, respectively. (C) LEF1, Flag epitope-tagged SUMO1 or SUMO2 and T7-PIASy were transiently expressed in 293T cells. LEF1 proteins in total RIPA buffer cell lysates were detected by an anti-LEF1 immunoblot (top). The expression of T7-PIASy proteins in total cell lysates was determined by an anti-T7 immunoblot (bottom).

To provide further evidence for SUMO modification of LEF1 in the presence of PIASy, we transfected gene constructs encoding LEF1 or T7-PIASy, together with a construct encoding Flag-tagged SUMO1 or Flag-tagged SUMO2. Immunoblot analysis of LEF1 proteins that were coimmunoprecipitated with anti-T7 antibody allowed for detection of an 18-kD-larger, slower-migrating form of LEF1 (Fig. ​(Fig.2B,2B, lane 3). In the presence of Flag-SUMO2, two slower-migrating forms of LEF1 were detected (Fig. ​(Fig.2B,2B, lane 5). The relative increase in the molecular mass of the largest form of LEF1 is consistent with the addition of the Flag epitope to SUMO2. This slower-migrating form of LEF1 is less abundant in the presence of Flag-SUMO1 (Fig. ​(Fig.2B,2B, lane 4), which might reflect a lower efficiency of conjugation with SUMO1 relative to SUMO2 and/or a lower extraction efficiency of SUMO1-conjugated LEF1 under our coimmunoprecipitation conditions. To distinguish between these two possibilities, we examined the sumoylation status of LEF1 in the absence or presence of Flag-SUMO1 or Flag-SUMO2 in total cell lysates harvested in RIPA buffer (Fig. ​(Fig.2C).2C). Under these stringent conditions, we could detect LEF1 modified by endogenous SUMO and by ectopically expressed Flag-SUMO1 and Flag-SUMO2. Intriguingly, expression of T7-PIASy enhanced SUMO modification of LEF1 with endogenous SUMO (cf. lanes 2 and 5) and with Flag-SUMO2 (cf. lanes 4 and 7), but it did not enhance SUMO modification of LEF1 with Flag-SUMO1 (cf. lanes 3 and 6). Taken together, these results suggest that PIASy preferentially augments modification of LEF1 with SUMO2.

The anti-SUMO2/3 immunoblot analysis of proteins that had been coimmunoprecipitated with antibodies directed against T7-PIASy suggested that PIASy is involved in the sumoylation of many proteins. To confirm the general role of PIASy in sumoylation of proteins in vivo and to examine the function of structural motifs in PIASy, we coexpressed wild-type or mutant forms of PIASy together with Flag-SUMO1 or Flag-SUMO2. As the RING motif has been implicated as an important functional determinant of ubiquitin E3 ligases (Jackson et al. 2000; Joazeiro and Weissman 2000), we examined the effects of mutations of Cys 330, 335, and 340 and His 337 in the RING domain of PIASy. In addition, we mutated Ser 470–474 in the serine-rich and acidic domain of PIASy, which bears similarity with a SUMO interaction motif in the PM-Sc175 protein (Minty et al. 2000). Immunoblot analysis of total cell lysates indicated that overexpression of wild-type PIASy increased the accumulation of Flag-SUMO–conjugated proteins relative to cells that had been transfected with the Flag-SUMO constructs alone (Fig. ​(Fig.3A,3A, cf. lanes 2,3 and lanes 5,6). The mutations of the Cys/His residues in the RING domain markedly decreased the sumoylation of endogenous proteins (lanes 7,8), whereas the Ser mutations had only a modest effect (lanes 9,10). Immunoblot analysis with anti-T7 antibodies confirmed the similar expression of wild-type and mutant PIASy proteins (Fig. ​(Fig.3A,3A, lower panel).

Open in a separate window

Figure 3

PIASy enhances SUMO-conjugation in vivo. (A) Wild-type PIASy enhances, whereas mutant PIASy proteins abrogate, SUMO-conjugation. Wild-type, RING mutant, or Ser mutant T7-PIASy proteins were coexpressed with Flag-SUMO1 or Flag-SUMO2 in 293T cells. The RING mutant contains serine substitutions for cysteines 330, 335, and 340 and an alanine substitution for histidine 337 in the RING domain. The Ser mutant contains alanine substitutions for serines 470 to 474 in the Ser/Ac domain. Flag-SUMO–modified proteins from total sample buffer cell lysates were detected by an anti-Flag immunoblot (top). Numbers to the right indicate the molecular mass of protein markers in kilodaltons. The expression of T7-PIASy proteins in total cell lysates was determined by an anti-T7 immunoblot (bottom). (B) Characterization of mutant PIASy and LEF1 proteins. Wild-type or mutant LEF1 and T7-PIASy proteins were transiently expressed in 293T cells. Equivalent amounts of total cellular protein were immunoprecipitated with anti-T7 mAb. Coimmunoprecipitated LEF1 proteins were detected by an anti-LEF1 immunoblot (top). The LEF1 M2/K267R protein contains alanine substitutions for lysine 25, aspartate 26, and glutamate 27 (Hsu et al. 1998) and an arginine substitution for lysine 267 within the two consensus SUMO motifs. The expression of LEF1 or T7-PIASy proteins in total cell lysates was determined by anti-LEF1 (middle) or anti-T7 (bottom) immunoblots, respectively.

Finally, we sought to identify the lysine residue(s) in LEF1 that are conjugated with SUMO proteins. We searched for the presence of consensus SUMO modification sites (ψKXE, in which ψ is a large hydrophobic residue, and K is the lysine to which SUMO is conjugated; Rodriguez et al. 2001; Sampson et al. 2001) in LEF1 and identified two putative sites at lysine residues 25 (FKDE) and 267 (VKQE). We generated mutations in both lysines (M2/K267R) and examined their effects on sumoylation of LEF1 in the presence of PIASy. The slower-migrating form of LEF1, corresponding to SUMO-modified LEF1, was not detected, suggesting that the mutated sequences represent bona fide sumoylation sites (Fig. ​(Fig.3B,3B, cf. lanes 2 and 5). Mutation of K267R alone decreased, but did not abrogate, the amount of coimmunoprecipitated SUMO-LEF1, suggesting that both sites in LEF1 can be conjugated with SUMO (data not shown). Mutations of the RING domain in PIASy abrogated SUMO modification of LEF1 and slightly reduced association with LEF1 (Fig. ​(Fig.3B,3B, lane 3; data not shown). In contrast, the Ser mutations did not abrogate SUMO modification or association with LEF1 (Fig. ​(Fig.3B,3B, lane 4). The relative levels of LEF1 and PIASy expression were determined by immunoblot analysis of total cell lysates (Fig. ​(Fig.3B,3B, lower panels). Taken together, these experiments suggest that the expression of PIASy results in extensive SUMO conjugation of multiple proteins, including LEF1. Moreover, the experiments indicate that the RING domain is an important structural determinant that is required for SUMO conjugation.

PIASy stimulates SUMO conjugation of LEF1 in a reconstituted system in vitro

The stimulatory effect of PIASy on the sumoylation of proteins raises the question as to whether PIASy is directly involved in this process. The conjugation of SUMO to target proteins shares many similarities with the ubiquitin system, although no E3 ligase for SUMO-modified proteins has yet been identified. To examine whether LEF1 can be sumoylated in the presence of E1 and E2 enzymes alone or whether PIASy facilitates this process, we used an in vitro reconstituted system with purified proteins. Incubation of recombinant LEF1 with SUMO1, the E1 enzyme Aos1/Uba2, the E2 enzyme Ubc9, and ATP did not result in significant SUMO conjugation of LEF1 (Fig. ​(Fig.4A,4A, lanes 2,14). However, addition of a precipitated T7-PIASy immune complex or bacterially expressed GST-PIASy allowed for efficient and multiple conjugation of SUMO1 to LEF1 (Fig. ​(Fig.4A,4A, lanes 3,15,16). Conjugation of SUMO1 was dependent on all other components and was not detected with PIASy protein in which the RING domain had been mutated (Fig. ​(Fig.4A,4A, lane 4). The slower-migrating forms of LEF1 were identified as SUMO1 conjugates by immunoblot analysis with an anti-SUMO1 antibody (data not shown). PIASy also augmented the conjugation of SUMO2 to recombinant LEF1 in vitro (Fig. ​(Fig.4A,4A, lane 12). The purity of the T7-PIASy immune complexes was examined on a silver-stained gel, showing that the T7-PIASy proteins were the predominant proteins in the immune complexes, and stoichiometric levels of additional copurifying proteins could not be detected (Fig. ​(Fig.4B).4B). Taken together, these data indicate that PIASy can function as a SUMO E3 ligase for LEF1. Thus, the enzymatic requirements of SUMO modification are similar to those of the ubiquitin modification process.

Open in a separate window

Figure 4

PIASy functions as a SUMO E3 ligase for LEF1 in vitro. (A) PIASy stimulates SUMO conjugation of LEF1. Recombinant LEF1 (25 ng) was incubated in the absence or presence of 5 mM ATP, 60 ng recombinant SUMO1 or SUMO2, 150 ng of recombinant E1 (Aos1/Uba2 heterodimer), 10 ng of recombinant E2 (Ubc9), and 4 μL of immune complex (IC)–purified wild-type (wt), RING mutant, or mock-transfected T7-PIASy for 60 min at 30°C, as indicated. (Lanes 14–16) Bacterially expressed wild-type GST-PIASy (300 or 1000 ng) was used instead of IC-purified T7-PIASy, as indicated. Reactions were terminated by the addition of sample buffer. LEF1 and SUMO-modified LEF1 were resolved by SDS-PAGE and detected by anti-LEF1 immunoblots. SUMO-modified LEF1 in lanes 15 and 16 migrates slower than in lanes 3 and 12 because of different electrophoresis conditions. (B) IC-purified T7-PIASy proteins were analyzed by SDS-PAGE and detected by silver staining to determine their purity and relative abundance. (C) SUMO-modified LEF1 binds DNA. Recombinant LEF1 either was sumoylated in the presence of ATP, E1, E2, SUMO1, and wild-type PIASy as described above (SUMO1-LEF1) or was treated under the same conditions without the E1 enzyme (ΔE1), so that LEF1 did not become sumoylated. The extent of LEF1 sumoylation was determined by an anti-LEF1 immunoblot using equivalent amounts of the ΔE1 and sumoylation reactions (D, input). The ability of unmodified LEF1 (25 ng) or sumoylated LEF1 (25 ng) to bind to a wt or mutated (mut) LEF1 site was determined by electrophoretic mobility shift assays. (D) SUMO-modified LEF1 binds to β-catenin. The ability of equivalent amounts of unmodified or SUMO-modified LEF1 to bind to immobilized His₆–β-catenin was analyzed by an in vitro association assay. The input, unbound, and His₆–β-catenin–bound LEF1 was detected by an anti-LEF1 immunoblot. The recombinant LEF1 proteins did not bind to nickel beads alone (data not shown).

In LEF1, two consensus sites for SUMO conjugation were identified, one within the β-catenin interaction domain at position 25 and one immediately upstream of the HMG domain at position 267. To examine whether SUMO modifications of LEF1 alter the DNA-binding ability and/or the association with β-catenin, we generated SUMO1-modified LEF1 in vitro and performed an electrophoretic mobility shift assay with a radiolabeled LEF1-binding site. Similar DNA binding was detected with LEF1 and SUMO1-modified LEF1 (Fig. ​(Fig.4C).4C). In addition, we used LEF1 and SUMO1-modified LEF1 in pull-down experiments with immobilized His-tagged β-catenin, and we visualized the bound LEF1 proteins by immunoblot analysis with anti-LEF1 antiserum. Both LEF1 and SUMO-LEF1 associated with β-catenin with similar efficiency (Fig. ​(Fig.4D).4D). Together, these experiments indicate that PIASy functions as a SUMO E3 ligase for LEF1 and that SUMO modification of LEF1 does not alter its interactions with DNA and β-catenin.

Repression of LEF1 activity by PIASy

The function of LEF1 as a transcriptional regulator can be positively or negatively modulated through association with additional cofactors. In response to Wnt signaling, the association of LEF1 with β-catenin results in a marked increase in transcriptional activation (van de Wetering et al. 1997; Hsu et al. 1998). Coexpression of LEF1 and PIASy in transfected 293T cells, which lack appreciable levels of endogenous LEF1, did not result in any detectable activation of a LEF₇-fos-luciferase reporter construct containing multiple LEF1-binding sites (data not shown). However, PIASy markedly reduced transcriptional activation of the reporter construct by LEF1 and β-catenin in a dose-dependent manner, but it did not reduce the basal activity of a reporter construct lacking the LEF1-binding sites (Fig. ​(Fig.5A;5A; data not shown). Likewise, PIASy efficiently antagonized the activation of the reporter gene by TCF1, another LEF1/TCF family member, and β-catenin (data not shown). We also observed the repressive effects of PIASy with a reporter construct in which the natural Twin promoter was linked to the luciferase gene (Fig. ​(Fig.5B;5B; Nishita et al. 2000). This PIASy-mediated repression was not observed with a mutated Twin promoter lacking the LEF1-binding sites (Fig. ​(Fig.5B).5B). To determine whether PIASy antagonizes the activation potential of LEF1 in the absence of β-catenin, we used a TCRα-fos-CAT reporter construct, which is stimulated by LEF1 in combination with Ets1 and AML1 (Fig. ​(Fig.5C).5C). PIASy repressed the activity of the TCRα enhancer, suggesting that PIASy antagonizes both Wnt-dependent and Wnt-independent activation by LEF1. We also confirmed that PIASy represses the activity of endogenous LEF1 in transfections of Jurkat T cells with the LEF₇-fos-Luc reporter construct and β-catenin (Fig. ​(Fig.5D).5D). Intriguingly, a mutant form of PIASy, in which the RING motif had been mutated, increased transcriptional activation by a factor of two, suggesting that this mutation generates a dominant-negative form of PIASy (Fig. ​(Fig.5D).5D). Furthermore, the ability of the RING mutant to activate LEF1-dependent transcriptional activation suggests that endogenous PIASy might normally repress LEF1 activity. Mutation of the Ser-rich domain of PIASy only modestly reduced the extent of repression relative to wild-type PIASy (Fig. ​(Fig.5D).5D). Finally, we examined the contribution of the two SUMO conjugation sites in LEF1 for PIASy-mediated repression. PIASy repressed the transcriptional activation potential of LEF1 M2/K267R almost as efficiently as that of wild-type LEF1 (Fig. ​(Fig.5E).5E). Thus, the repression of LEF1 activity by PIASy requires the RING domain of PIASy, but it does not require the two consensus sumoylation sites in LEF1.

Open in a separate window

Figure 5

PIASy represses LEF1 activity. (A) PIASy represses LEF1 activity from a multimerized LEF1 reporter. 293T cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (25 ng; for normalization), LEF1 (30 ng), β-catenin (1 μg), or increasing amounts of T7-PIASy (0.1, 0.3, or 1 μg), as indicated. For this and subsequent experiments, the levels of luciferase or CAT activity were normalized for β-galactosidase activity and are expressed as fold activation relative to the level of luciferase or CAT activity from cells transfected with the reporter construct alone. All of the transfection experiments were performed at least three times, and the results of representative experiments are shown. (B) PIASy represses LEF1-dependent Twn reporter activity. NMuMG epithelial cells were transfected with 2 μg of the Twn luciferase reporter construct containing either wild-type (Twn-Luc) or mutated (ΔLEF-Twn-Luc) LEF1-binding sites together with expression constructs encoding for β-galactosidase (0.5 μg; for normalization), LEF1 (0.5 μg), β-catenin (5 μg), or PIASy (1 μg), as indicated. (C) PIASy represses TCRα enhancer activity. 293T cells were transfected with 0.25 μg of a TCRα-CAT reporter construct together with expression constructs encoding for β-galactosidase (50 ng; for normalization), LEF1 (100 ng), Ets1, and AML1 (250 ng each) or T7-PIASy (0.3 or 1 μg), as indicated. (D) Wild-type PIASy represses endogenous LEF1 activity. Jurkat cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (0.5 μg; for normalization), β-catenin (5 μg), or increasing amounts of wild-type T7-PIASy (1 or 3 μg), RING-mutated T7-PIASy (1 or 3 μg), or Ser-mutated T7-PIASy (1 or 3 μg), as indicated. (E) Mutation of the SUMO consensus sites in LEF1 does not abrogate PIASy-mediated repression of LEF1 activity. 293T cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (25 ng; for normalization), wild-type or mutant M2/K267R LEF1 (30 ng), β-catenin (1 μg), or increasing amounts of T7-PIASy (0.1, 0.3, or 1 μg), as indicated.

PIASy binds to nuclear matrix attachment region DNA

The N-terminal domain of PIASy, which is involved in interaction with LEF1, has sequence similarity with the nuclear matrix–binding protein SAF-A (Kipp et al. 2000). The region of homology between PIAS proteins and SAF-A has been termed the SAP domain and has been proposed to mediate binding to DNA sequences that are found in nuclear matrix attachment regions (MARs; Aravind and Koonin 2000). To examine the putative binding of PIASy to nuclear matrix–associated DNA, we performed an electrophoretic mobility shift assay with purified GST-PIASyN97 and radiolabeled oligonucleotides encompassing a wild-type or mutated MAR consensus sequence (Bode et al. 1992). Binding was detected with the wild-type MAR sequence but was significantly diminished with a mutant MAR sequence or a LEF1-binding site oligonucleotide (Fig. ​(Fig.6A;6A; data not shown).

Open in a separate window

Figure 6

PIASy binds to nuclear matrix attachment region (MAR) DNA. (A) The PIASy SAP domain binds to MAR DNA. A GST-PIASyN97 recombinant fusion protein (0.1 or 0.3 μg) encompassing the putative SAP domain was examined for its ability to bind to a wild-type (wt) or mutated (mut) MAR consensus sequence by electrophoretic mobility shift assays, as indicated. (B) The N-terminal SAP domain of PIASy is required for nuclear matrix association in vivo. COS7 cells were transiently transfected with expression vectors encoding for wt T7-PIASy, a mutant T7-PIASy protein lacking its N-terminal 93 amino acids and SAP domain but containing a heterologous NLS for nuclear targeting (T7-PIASy–Δ93-NLS), or protein A–tagged Bright. At 48 h posttransfection, the cells were either immediately fixed (upper panels) or were processed for nuclear matrix preparations before fixation (lower panels). T7 epitope-tagged PIASy proteins were detected by indirect immunofluorescence with an anti-T7 mAb. Bright, a protein known to associate with MARs and the nuclear matrix, was detected by indirect immunofluorescence with rabbit IgG. Images were collected by confocal microscopy. The cells shown are representative of >70% of transfected cells.

To confirm the role of the N terminus of PIASy in mediating the association with the nuclear matrix, we compared the retention of wild-type PIASy and a mutant form of PIASy lacking the N-terminal 93 residues encompassing the SAP domain, on nuclear matrix preparations. Because the deletion of the N terminus of PIASy impaired nuclear localization (data not shown), we added a heterologous nuclear localization sequence to the mutant protein. In these experiments, we used the MAR-binding protein Bright as a positive control (Zong et al. 2000). As anticipated, Bright was retained in the nuclear matrix preparation (Fig. ​(Fig.6B).6B). Likewise, PIASy, but not ΔN93-PIASy-NLS, was retained in the nuclear matrix preparation, suggesting that the N terminus of PIASy mediates the association with MARs and the nuclear matrix.

Role of SUMO conjugation of LEF1 and PIASy association

The expression of PIASy results in both sumoylation and subnuclear sequestration of LEF1. Although the physiological relevance of SUMO conjugation of LEF1 remains unclear, three lines of evidence suggest that SUMO modification of LEF1 augments its association with PIASy and targeting of LEF1 to nuclear bodies. First, the ratio of SUMO-conjugated LEF1 to unmodified LEF1 is markedly increased in coimmunoprecipitation assays with T7-PIASy relative to that observed in total cell lysates. The effect of sumoylation of LEF1 on its interaction with PIASy is reminiscent of the enhancement of the association between Sp100 and HP1 by SUMO conjugation of Sp100 (Seeler et al. 1998, 2001). Second, coexpression of SUMO1 or SUMO2 with LEF1 and PIASy enhances the targeting of LEF1 to nuclear bodies. Third, the RING mutant PIASy protein, which is deficient for SUMO conjugation, is unable to sequester LEF1 in nuclear bodies and functions as a dominant-negative protein in transcriptional activation assays. These results suggest that SUMO modification, nuclear body sequestration, and transcriptional repression are closely linked. Although the analysis of the LEF1 M2/K267R mutant indicates that sumoylation of LEF1 may not be a prerequisite for its association with PIASy and its subnuclear sequestration, SUMO modification of another critical target in the PIASy pathway might be required to inhibit transcription independent of SUMO modification of LEF1. Alternatively, LEF1 might be sumoylated at low levels at additional, nonconsensus SUMO conjugation sites, which cannot be easily detected in Western blots but might be sufficient for subnuclear sequestration and repression of LEF1 activity.

The function of SUMO conjugation is, with a few exceptions, still fairly obscure (for review, see Melchior 2000; Muller et al. 2001). Similar to LEF1, the p53 protein is sumoylated and mutation of the major SUMO conjugation site, K386R, does not affect p53-dependent transactivation, growth suppression, or targeting of p53 to nuclear bodies (Fogal et al. 2000; Kwek et al. 2001). However, coexpression of p53 with SUMO1 and Ubc9 or with PML3 can enhance the recruitment of p53 to nuclear bodies (Fogal et al. 2000). Thus, the recruitment of LEF1 and p53 to subnuclear structures might be the critical determinant for the regulation of these proteins, and the sumoylation may simply be a consequence of the sequestration.

PIASy-mediated targeting of LEF1 to nuclear bodies correlates well with the repression of LEF1 activity. PIASy itself is localized in nuclear bodies and the nuclear matrix, and this localization depends on the presence of the N-terminal SAP domain. The subnuclear localization of LEF1 in the presence of PIASy overlaps with a subset of PML nuclear bodies. These subnuclear structures are quite heterogenous and have been implicated in transcriptional repression, transcriptional activation, and protein degradation (for review, see Hatta and Fukamizu 2001). The protein half-life of LEF1 does not significantly change in the presence of PIASy (t_1/2 = 13 h without PIASy versus t_1/2 = 10.2 h with PIASy; data not shown). Therefore, the activity of LEF1 may be repressed by targeting of LEF1 to heterochromatin or by recruitment of LEF1 into complexes containing corepressors. For example, targeting of the transcription factor Ikaros to heterochromatin has been shown to mediate the repression of Ikaros target genes (Brown et al. 1997). However, nuclear bodies containing LEF1 and PIASy do not colocalize with dimethylated histones, a hallmark of heterochromatin (Nielsen et al. 2001). PML nuclear bodies have been found to contain transcriptional corepressors, such as Smrt/N-CoR, and histone deacetylases (HDACs). Treatment of cells with trichostatin A, an inhibitor of HDACs (for review, see Yoshida and Horinouchi 1999), did not antagonize PIASy-mediated repression of LEF1 activity (data not shown). Therefore, PML components different from HDACs likely account for PIASy-mediated repression of LEF1 activity. All PIAS family members contain a LXXLL signature motif within the SAP domain, which is required for transrepression and may be recognized by as yet unidentified corepressor proteins (Liu et al. 2001).

Protein expression and purification

The ³⁵S-labeled full-length and truncated LEF1 proteins were in vitro translated from pBluescript KS+ (Stratagene) using rabbit reticulocyte lysate (Promega). For the bacterially expressed proteins, purification involved IPTG-induced expression in Escherichia coli BL21 gold (Stratagene). Buffers contained 1 μg/mL each of leupeptin, pepstatin, and aprotinin and 1 mM DTT (or β-mercaptoethanol). Lysis buffers also contained 0.1 mM PMSF. For SUMO1 purification, bacteria lysed in 50 mM Tris-Cl (pH 8.0) and 50 mM NaCl were precleared with Q Sepharose (Sigma) and purified by gel filtration. For purification of catalytically active SUMO E1 enzyme, His-Aos1 and Uba2 were coexpressed in bacteria; lysed in 50 mM Na-phosphate buffer (pH 8.0), 300 mM NaCl, and 10 mM imidazole; and purified on ProBond Resin (Invitrogen), followed by molecular sieving (Superdex 200) and ion exchange chromatography (Mono Q, Pharmacia Biotech). For purification of Ubc9, bacteria were lysed in 50 mM Na-phosphate buffer (pH 6.5) and 50 mM NaCl, incubated with SP-Sepharose beads (Sigma), eluted with 50 mM Na-phosphate buffer (pH 6.5) and 300 mM NaCl, and sieved through a Superdex 200 column. All of the proteins were dialyzed against transport buffer (20 mM HEPES at pH 7.5, 110 mM K-acetate, 2 mM Mg-acetate, and 0.5 mM EGTA) before their use in the in vitro sumoylation assays. For purification of GST-PIASyN97 or full-length GST-PIASy, bacteria were lysed in 10 mM Na-phosphate buffer (pH 7.2), 150 mM NaCl, 10 mM EDTA, 2 mM DTT, and protease inhibitor mix (5 μg/mL each of trypsin/chymotrypsin inhibitor, antipain, aprotinin, bestatin, and leupeptin; 0.25 μg/mL pepstatin; and 1 mM PMSF; Sigma) and purified on glutathione-agarose beads according to the manufacturer's instructions (Pharmacia). For purification of His₆–β-catenin, bacteria were lysed in 20 mM HEPES (pH 7.5), 300 mM NaCl, 0.2% TX-100, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitor mix and purified on Ni-NTA beads according to the manufacturer's protocol (Qiagen). Protein concentrations were determined by Bradford assay according to the manufacturer's protocol (Bio-Rad).

In vitro sumoylation assays

For immune complex (IC) purification of T7-PIASy proteins, 293T cells were either mock transfected or were transfected with expression constructs encoding for wild-type T7-PIASy or T7-PIASy RING mutant. At 48 h posttransfection, cells were harvested in WL buffer (50 mM Tris-Cl at pH 8.0, 400 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM EDTA, 1 mM DTT, protease inhibitor mix, 1 mM NaF, and 0.4 mM Na-orthovanadate). Clarified lysates (∼4 mg total cellular protein) were diluted fivefold in WL buffer lacking NaCl, and the T7-PIASy proteins were immunoprecipitated with 5 μg of anti-T7 mAb and 200 μL of a 50% slurry of protein G-Sepharose as described (Harlow and Lane 1988). The ICs were washed 4 times with WL buffer containing 400 mM NaCl and washed 2 times with transport buffer. The sedimented ICs were resuspended in an equal volume of transport buffer. The purity and relative abundance of the sedimented T7-PIASy ICs was determined by SDS-PAGE and silver staining and by anti-T7 immunoblots. The sumoylation assays with recombinant proteins were performed in a total volume of 20 μL in transport buffer supplemented with 0.05% Tween 20, 0.2 mg/mL ovalbumin grade VI, and 1 mM each aprotinin, leupeptin, pepstatin, AEBSF, and DTT (Sigma). Reactions were incubated for 1 h at 30°C and stopped by the addition of SDS sample buffer. The samples were resolved on a 7% polyacrylamide gel, and sumoylated LEF1 was detected by an anti-LEF1 immunoblot. Concentrations of recombinant proteins were as indicated in the figure legends.

Cell culture, transient transfections, and reporter assays

293T and COS7 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with penicillin-streptomycin-glutamine (PSG) and 10% fetal bovine serum (FBS; Invitrogen Life Technologies). NMuMG cells were cultured in DMEM supplemented with PSG, 10 μg/mL insulin (Sigma), and 10% FBS. Jurkat T cells were cultured in RPMI 1640 supplemented with PSG, 50 μM β-mercaptoethanol (Sigma), and 10% FBS. 293T, COS7, and NMuMG cells were plated onto 60-mm tissue culture plates 12 h before transfection and were transfected with 10 μg of plasmid DNA by the calcium phosphate method (Sambrook et al. 1989). Jurkat cell transfections were performed by electroporation with 30 μg of plasmid DNA as described (Hughes and Pober 1996). The total DNA concentration in each transfection experiment was kept constant by adding vector plasmid DNA. Cells were harvested 36 to 48 h posttransfection in 200 μL of reporter lysis buffer, and luciferase assays were conducted according to the manufacturer's instructions (Promega). CAT and β-galactosidase assays were performed as described (Starr et al. 1996).

In vitro association assays, immunoprecipitation, and Western blot analysis

In vitro GST-PIASyN97 and His₆–β-catenin pull-down assays were conducted essentially as described (Bruhn et al. 1997). For the in vitro His₆–β-catenin association assays, recombinant LEF1 or SUMO-LEF1 was dialyzed against 10 mM Tris-Cl (pH 8.0), 50 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor mix before use in the pull-down assays. For coimmunoprecipitation experiments, cells were harvested in Coimmunoprecipitation buffer (20 mM Tris-Cl at pH 8.0, 25 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% TX-100, 10% glycerol, 1 mM DTT, protease inhibitor mix, 1 mM NaF, and 0.4 mM Na-orthovanadate). Equivalent amounts of total protein were precleared and immunoprecipitated with 2 μg of anti-T7 mAb (Novagen) as described (Harlow and Lane 1988). For direct immunoprecipitation of target proteins, cells were harvested in RIPA buffer (10 mM Na-phosphate buffer at pH 7.2, 150 mM NaCl, 1% Na-deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM DTT, protease inhibitor mix, 1 mM NaF, and 0.4 mM Na-orthovanadate). Western blots were conducted by the ECL method according to the manufacturer's instructions (Amersham-Pharmacia), with the following antibodies and dilutions: rabbit anti-LEF1 antiserum, 1:4000; anti-T7 mAb (Novagen), 1:5000; and anti-Flag M2 mAb (Sigma), 1:4000.

Electrophoretic mobility shift assay

The wild-type MAR (5′-TCTTTAATTTCTAATATATT TAGAATTC-3′) or mutant MAR (5′-TCTTTAATTTCTACT GCTTTAGAATTC-3′) oligonucleotides were radiolabeled with ³²P and gel purified on a 20% native polyacrylamide gel. The LEF1 oligonucleotides have been previously described (Hsu et al. 1998). DNA-binding reactions contained 20 mM HEPES (pH 7.9), 50 mM NaCl, 1 mM DTT, 5 mM EDTA, 5% glycerol, 2 μg of BSA, 10 ng of poly dI-dC, 5000 cpm of radiolabeled probe, and the amount of protein indicated in the figure legends. Electrophoretic mobility shift assays were conducted essentially as described (Bruhn et al. 1997; Hsu et al. 1998).

We thank Dr. Juan Galceran for expert assistance in preparation of the figures and for helpful suggestions on the manuscript, Gergana Dobreva for the cloning of ΔN93-PIASy-NLS and assistance with nuclear matrix preparations, Dr. Walter Muranyi for assistance with confocal microscopy, and members of the Grosschedl lab for insightful discussions. We thank Dr. Thomas Jenuwein for anti-dimethyl lysine 9 H3 antiserum, Dr. Hisato Saitoh for anti-SUMO2/3 antiserum, Dr. Stefan Stamm for anti-SC35 mAb, Dr. Ken Cho for the Twin luciferase reporter constructs, Dr. Thomas Stamminger for the Flag-SUMO1 and -SUMO2 expression constructs, and Dr. Philip Tucker for the Bright and HA-Sp100 expression constructs. S.S. is a fellow of the Leukemia and Lymphoma Society of America. This work was supported by funds from the German Research Foundation (DFG).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.