PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling Wera Roth, Claudio Sustmann, Matthias Kieslinger, Andrea Gilmozzi, Denis Irmer, Elisabeth Kremmer, Chris Turck and This information is current as Rudolf Grosschedl of October 2, 2021. J Immunol 2004; 173:6189-6199; ; doi: 10.4049/jimmunol.173.10.6189 http://www.jimmunol.org/content/173/10/6189 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling1

Wera Roth,* Claudio Sustmann,* Matthias Kieslinger,* Andrea Gilmozzi,* Denis Irmer,* Elisabeth Kremmer,† Chris Turck,‡ and Rudolf Grosschedl2*

Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-␥ and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target . Two-dimensional analysis of total Downloaded from proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins. The Journal of Immunology, 2004, 173: 6189–6199.

rotein inhibitors of activated STATs (PIAS)3 have been with other proteins. PIAS1 and PIAS3 inhibit DNA binding of identified as interaction partners for multiple nuclear pro- STAT1 and STAT3, respectively (1, 2). In contrast, PIASy represses P teins, including the STATs and lymphoid enhancer bind- STAT1, LEF1, Smad4 and the AR without interfering with DNA http://www.jimmunol.org/ ing factor (LEF)1/T cell-specific factor (TCF) proteins that medi- binding by these proteins (3, 24, 26, 27). The Piasx gene encodes two ate nuclear responses to and Wnt signals, respectively splice variants, x␣ (ARIP3) and x␤ (Miz1), which interact with the (1–5). The cytokines IFN-␣ and IFN-␥ are the main cytokines for AR and with the homeodomain protein Msx2, respectively (28, 29). innate immune responses against viral infections. IFN-␣ is pro- In addition, PIASx␤ interacts with STAT4, following IL-12 stimula- duced by many cell types, whereas IFN-␥ is produced predomi- tion of T cells (5). In most cases, the association of PIAS proteins with nately by hemopoietic cells (reviewed in Refs. 6 and 7). The sig- transcription factors results in repression (reviewed in Ref. 30); how- naling pathways for both IFNs are similar and involve the Jak ever, PIAS proteins can also augment by synergizing tyrosine kinase-mediated activation of STAT proteins that stimu- with other transcriptional coactivators (4, 18). by guest on October 2, 2021 late transcription of target genes alone or in concert with IFN reg- PIAS proteins share a similar domain structure, including an ulatory factors (reviewed in Refs. 8 and 9). The response of cells N-terminal domain, termed SAP domain, which mediates, in part, to Wnt signals involves the stabilization and nuclear translocation the interaction with partner proteins (3, 31) and a central RING ␤ of -catenin, which associates with LEF/TCF proteins and acti- domain that resembles the catalytic domain of ubiquitin ligases vates Wnt-responsive genes (reviewed in Ref. 10). (32, 33). Recent experiments aimed at the mechanisms by which In addition to STATs and LEF1/TCF proteins, many other tran- PIAS proteins modulate the activity of their interaction partners, scription factors interact with PIAS proteins. These include nuclear have shown that PIAS proteins can augment the covalent modifi- hormone receptors, such as the androgen receptor (AR), p53, cation of proteins with the small ubiquitin-related modifier Smad4, Sp3, HMGI-C, Gfi-1, IRF-1, TFII-I and yeast septins (11– (SUMO) (3, 14–16, 22, 34–36). Two families of SUMO proteins, 25). In mouse and man, four Pias genes (Pias1, Pias3, Piasx, and SUMO-1 and SUMO-2/SUMO-3 resemble ubiquitin proteins and Piasy) have been identified, which encode proteins that share a are also attached to the ⑀-amino groups of lysines in target proteins similar domain structure but differ in their specificity of interaction (37). The sumoylation pathway resembles that of ubiquitination, with the activation of SUMO by an E1 protein and its transfer to an E2 (37–39). Multiple lines of evidence suggest that *Gene Center and Institute of Biochemistry, University of Munich, †GSF National Research Center for Environment and Health, and ‡Max-Planck-Institute of Psychi- PIAS proteins act as E3 ligases for SUMO. First, PIAS proteins atry, Munich, Germany can cooperate with E1 and E2 in reconstituted systems to Received for publication May 21, 2004. Accepted for publication August 20, 2004. augment SUMO modification of target proteins (3, 14, 22, 35). The costs of publication of this article were defrayed in part by the payment of page Second, PIAS proteins contain a cysteine-rich RING domain, charges. This article must therefore be hereby marked advertisement in accordance which is a hallmark for ubiquitin ligases and is functionally im- with 18 U.S.C. Section 1734 solely to indicate this fact. portant for the sumoylation activity of PIAS proteins (3, 22, 33– 1 This work was supported by a Grant of the German Research Foundation TR-5. 35). Finally, PIAS proteins display some specificity of protein sub- 2 Address correspondence and reprint requests to Dr. Rudolf Grosschedl at the current strates, although the specificity is not as pronounced as for address: Max-Planck-Institute of Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany. E-mail address: [email protected] ubiquitin ligases (5, 22, 40). 3 Abbreviations used in this paper: PIAS, protein inhibitor of activated STAT; LEF, The function of PIAS proteins as SUMO E3 ligases appears lymphoid enhancer binding factor; TCF, T cell-specific factor; SUMO, small ubiq- to involve multiple mechanisms. The repression of transcription uitin-like modifier; ES, embryonic stem; IRF, IFN regulatory factor; AR, androgen factors by PIAS proteins often correlates with the subnuclear se- receptor; ␤-Gal, ␤-galactosidase; X-Gal, 5-bromo-4-chloro-3-indolyl ␤-D-galacto- side; MuLV, murine leukemia virus; MEF, mouse embryonic fibroblast. questration into speckles, such as promyelocytic leukemia nuclear

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6190 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO bodies (3, 20, 21, 23, 34, 41). This observation could account for and hybridized overnight at 42°C. Blots were washed to final stringency of the repression of transcription factor activity without changes in 0.2% SDS, 0.1% SDS at 65°C, and exposed to film. ␤ DNA binding. In addition, PIASx has been found to interact with Immunoblot analysis histone deacetylase-3, which is involved in repression of TFII-I family proteins (25). The activation function of PIAS proteins Total protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose. Immunoblots were conducted by the ECL method ac- could also be accounted for by the sumoylation-dependent aug- cording to the manufacturer’s instructions (Amersham Pharmacia Biotech, mentation of protein-protein interactions. In nuclear hormone re- Piscataway, NJ), with the Abs rat anti-PIASy (mouse) mAb. ceptors, the SUMO modification sites coincide with the protein motifs that mediate synergy in the activation of reporters contain- Flow cytometry ing multimerized binding sites for the hormone receptors (42–44). Thymus, spleen, and bone marrow were isolated and dispersed in RPMI Genetic experiments in Drosophila indicated that the PIAS or- 1640, containing antibiotics and glutamine with 10% heat-inactivated FCS. ϫ 6 ␮ thologue dPIAS, also known as Zimp, interacts functionally with Cells (1 10 ) were resuspended in 100 l of FACS buffer (PBS, 1% FCS) and incubated with Ab for 20 min. Cells were washed three times the STAT orthologue stat92E to regulate blood cell and eye de- with FACS buffer, and the process was then repeated with secondary Abs velopment (45). An additional role of PIAS in the regulation of as necessary. After staining, cells were resuspended in 500 ␮l of FACS structure and function was inferred from the identi- buffer containing propidium iodine. Cells were then analyzed using a FACS- fication and characterization of dPIAS as a suppressor of position- Calibur (BD Biosciences, San Jose, CA). Data generated was interpreted effect-variegation, Su(var)2-10 (46). Likewise, a yeast orthologue using CellQuest Software (BD Biosciences). Data shown in the figures are gated for lymphocytes by size and complexity and for living cells by ex- of PIAS proteins, termed Siz1 was identified through its genetic cluding cells that uptake propidium iodine. Directly conjugated Abs spe- interaction with the condensing complex (47) and shown to act as cific for mouse CD4, CD8, Mac1, and streptavidin-conjugated PE, FITC, a SUMO E3 ligase for septin proteins (14, 16). However, the func- and allophycocyanin were obtained from Caltag Laboratories (San Fran- Downloaded from tion of PIAS proteins in the mouse has not yet been elucidated. We cisco, CA). Directly conjugated Abs specific for mouse B220, CD43, GR1, and IgM were obtained from BD Biosciences. report the generation and analysis of a mouse carrying a targeted mutation of the Piasy gene. We find no gross phenotypic abnor- ␤-galactosidase (␤-Gal) detection of Piasy-␤-Gal expressing malities, although the Mendelian ratio of viable homozygous mu- lymphocytes tant offspring is significantly reduced relative to that of homozy-

Thymus, spleen, and bone marrow cell suspensions were prepared as de- http://www.jimmunol.org/ gous mutant embryos at E18.5. Molecular analysis of signaling by scribed above. After cell surface staining for flow cytometry, cells were IFN-␥ and Wnt proteins in PIASy-deficient mouse splenocytes and resuspended in growth medium. Cells were mixed with an equal volume of mouse embryonic fibroblasts (MEFs) revealed a modest but repro- 2 mM fluorescein-digalactoside (Molecular Probes, Eugene, OR), and in- ducible decrease in the nuclear responses, suggesting that PIASy cubated for 1 min at 37°C. Afterwards the cell suspensions were diluted 10-fold with ice-cold growth medium and incubated on ice for 30–60 min. acts redundantly with other PIAS proteins. Cells were centrifuged and resuspended in 500 ␮l of FACS buffer and run on a FACSCalibur (BD Biosciences). Materials and Methods X-Gal staining of whole mount embryos Generation of Piasy mutant mice Dissected E16.5 embryos were fixed with 4% paraformaldehyde in PBS for A mouse genomic library from 129/Sv mice (␭dashII) was screened with by guest on October 2, 2021 10 min on ice and stained for ␤-Gal activity by incubation in PBS con- a Piasy cDNA probe encompassing the full-length coding sequence of taining 2 mM MgCl ,5mMK Fe(CN) ,5mMK Fe(CN) , and 0.4 mg/ml mouse Piasy. A phage clone was isolated that included exons 2 through 9 2 4 6 3 6 5-bromo-4-chloro-3-indolyl ␤-D-galactoside (X-Gal) at 37°C for 2–24 h. of the mouse Piasy gene. A 10-kb genomic Piasy gene fragment (48), After staining, the embryos were washed in PBS, further fixed in 4% para- including exons 2 through 9, was excised by EcoRI and subcloned into a formaldehyde in PBS at 4°C and cleared using a benzyl alcohol to benzyl Bluescript vector (Stratagene, La Jolla CA). For the generation of a tar- benzoate ratio (1:2) according to standard procedures. geting construct a 2.1-kb genomic EcoRI/BamHI fragment, encompassing parts of intron 1, exon 1, and 217 bp of exon 2 was used in combination X-Gal staining of cryosections of mouse embryos with a 4.2-kb genomic, PCR-amplified fragment encompassing the 3Ј end of intron 2, exons 3 through 9 and the 5Ј end of intron 9. A LacZ-PGKneo E14.5 and E16.5 embryos were dissected in cold PBS and dry-frozen on gene cassette was inserted in frame into a BamHI site located in exon 2 dry ice. Prior cryostat sectioning, embryos were embedded and oriented in giving rise to the final targeting construct, where the 3Ј 188 bp of exon 2 tissue freezing medium (Jung; Leica, Deerfield IL). Twenty-micrometer and 5150 bp of intron 2 were deleted (see Fig. 1). To allow positive- cryosections were fixed for 15 min in 0.2% glutaraldehyde in fixing solu- negative selection, a HSV-tk expression cassette was inserted into the EagI tion (PBS, pH 7.4, containing 5 mM EGTA, pH 7.3, 2 mM MgCl2) at room site of the Bluescript vector. For electroporation of D3 embryonic stem temperature. After washing three times for 10 min in washing solution cells the targeting vector was linearized with SalI. Electroporation and (PBS, pH 7.4, containing 2 mM MgCl2, 0.02% Nonidet P-40), sections selection of embryonic stem (ES) cells and the processing of G418-resis- were stained overnight in X-Gal staining solution (washing solution, con- tant ES cell clones were performed as described (49). Homologous recom- taining 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 1 bination was identified by DNA blot analysis with internal, 5Ј and 3Ј ex- mg/ml X-Gal) at 37°C. Sections were counterstained for 75 s as indicated ternal probes (see Fig. 1 and data not shown). using Nuclear Fast Red (Vector Laboratories, Burlingame, CA) and mounted in DPX (Fluka, Buchs, Switzerland). Mice Plasmid constructs The animals used in all studies were maintained and bred according to institutional guidelines in the mouse facility of the Institute of Biochem- Plasmids encoding for full-length LEF1 have been previously described ␤ istry (University of Munich, Munich, Germany). A PCR-based screening (50, 51). The pLEF7-fos-luciferase vector and the pCMV- -catenin ex- method was used to distinguish wild-type, heterozygous, and homozygous pression vector have been previously described (52). The pRL-TK Renilla mutant mice using oligonucleotide primers as follows: 60–12c, 5Ј-GAT control vector (Promega, Madison, WI) was used for normalization in lu- GCTGCTGGGCTTTGTAG-3Ј; 60–10nc, 5Ј-CGTCCAGCATGTTGAA ciferase reporter assays. GAAG-3Ј; lac1–2, 5Ј-GTCGGATTCTCCGTGGGAA-3Ј. Cell culture, transient transfections, and reporter assays DNA blot analysis MEFs were harvested from E12.5 embryos according to standard proce- Genomic DNA was isolated from ES cells and tail biopsies. Then 15 ␮gof dures (53). Genotyping was performed as described. MEFs were cultured each sample was digested by restriction enzymes (see Fig. 1). The frag- in high glucose DMEM supplemented with penicillin-streptomycin-glu- ments were separated on a 0.8% agarose gel and transferred to nylon mem- tamine and 10% FCS (Invitrogen Life Technologies, Grand Island, NY). branes. The internal, the 5Ј and 3Ј external probes were labeled with MEFs were transfected using the Amaxa Nucleofactor (Amaxa Biosys- [␣-32P]dCTP by Random priming (Roche Diagnostics, Indianapolis, IN) tems, Cologne, Germany) and the MEF2 Nucleofactor kit (programme The Journal of Immunology 6191

A23). The total DNA concentration was kept constant by adding empty Results vector plasmid DNA. Cells were harvested 26–28 h posttransfection in 200 Targeted disruption of the Piasy gene ␮l of reporter lysis buffer, and luciferase assays were conducted according to the manufacturer’s instructions (Promega). Firefly luciferase activities With the aim of inactivating the murine Piasy gene in vivo, we were normalized against simultaneously measured Renilla luciferase ac- used a cloned 10-kb genomic Piasy DNA fragment to generate an tivity (Promega Dual Luciferase Reporter Assay System). insertion-type targeting construct, in which a LacZ-PGKneo gene Establishment of Abelson murine leukemia virus (MuLV) cassette has been inserted in-frame into the second exon of the transformed pre-B cell lines Piasy gene (Fig. 1A). Homologous recombination of the targeting Fetal livers were isolated from E16.5 embryos and dispersed in RPMI 1640 containing antibiotics and glutamine with 10% heat-inactivated FCS by standard techniques. The Abelson MuLV was used to immortalize pools of pre-B cell populations (54).

Splenocyte cultures Spleens were isolated from 3-mo-old mice and dispersed in RPMI 1640 containing antibiotics and glutamine with 10% heat-inactivated FCS by standard techniques. Cells were cultured overnight, counted, and 1.8 ϫ 107 cells each were replated at a density of 2.25 ϫ 106/ml. After 24 h, spleno- cytes were stimulated for 2 h with 500 U/ml murine IFN-␣ (R&D Systems, Minneapolis, MN) and 30 ng/ml murine IFN-␥ (R&D Systems). Spleno-

cytes were harvested by centrifugation followed by total RNA preparation Downloaded from using TRIzol (Invitrogen Life Technologies).

Bone marrow macrophage cultures Bone marrow was harvested from femurs of 3-mo-old animals by rinsing marrow cavities with PBS. Cells were cultured overnight in RPMI 1640 containing antibiotics and glutamine with 10% heat-inactivated FCS, and 10 ng/ml murine M-CSF (R&D Systems). Cells were counted and ϳ2.5 ϫ http://www.jimmunol.org/ 106 cells per plate were reseeded on new tissue culture dishes, and mac- rophages were expanded for 6 days using 10 ng/ml M-CSF. Expanded macrophages were stimulated for 2 h with 500 U/ml murine IFN-␣ (R&D Systems) and 30 ng/ml murine IFN-␥ (R&D Systems). Cells were har- vested by centrifugation followed by total RNA preparation using TRIzol (Invitrogen Life Technologies).

SYBR-green based real-time PCR The mRNA expression level of Wnt and STAT signaling target genes was measured using SYBR Green based real-time PCR. Total RNA was iso- by guest on October 2, 2021 ␮ lated with TRIzol (Invitrogen Life Technologies) and 2 g of RNA were FIGURE 1. Targeted inactivation of the murine Piasy gene. A, Targeted transcribed to cDNA using 200 U SuperscriptII (Invitrogen Life Technol- in-frame insertion of the bacterial lacZ gene into the murine Piasy locus ogies) and 0.1 nM oligo(dT) in a total volume of 20 ␮l. For one 12–18 (line) with the location of the exons (f) indicated. The 5Ј,3Ј, and internal real-time reaction, 2ϫ SYBR Green Mix (Applied Biosystems) was supple- mented with 1/40 of synthesized cDNA in addition to the appropriate oligo- probes are shown as bold bars. The targeting construct includes the lacZ nucleotide primer pair and run on the AbiPrism 7000 sequence detection coding sequence, fused in-frame with amino acid 81 of PIASy encoded by system (Applied Biosystems, Foster City, CA). Reverse transcriptase controls exon 2, in combination with a PGK-neor gene cassette ( ). The length of were done in parallel without adding enzyme. Obtained cycle numbers were the fragments generated by XhoI/HindIII (5Ј external probe), EcoRV (in- normalized to ␤-actin. All primer sequences were designed with the Primer ternal probe), and BamHI (3Ј external probe) restriction digests of the Express software (Applied Biosystems). The primer sequences and their final wild-type and mutant allele are indicated below. Representative restriction Ј concentrations used were as follows: Axin2 (forward, 600 nM), 5 - GAGGTG sites are indicated above the lines as: B, BamHI; H, HindIII; R, EcoRI; X, Ј Ј GTACCTTGCCAAAA-3 , Axin2 (reverse, 50 nM), 5 -TTCCTGTCCCT XhoI. B, Wild-type PIASy protein is shown schematically on the top. CTGCTGACT-3Ј, IFN regulatory factor (Irf)1 (forward, 600 nM), 5Ј- PIASy contains a putative chromatin-binding SAP (SAP) domain, a C HC CCCTGGCTAGAGATGCAGATTAAT-3Ј, Irf1 (reverse, 600 nM) 5Ј- 2 3 CATGGAATCTGGAAGATCATCTCTT-3Ј, ␤-actin (forward, 40 nM), RING (RING), a C-terminal serine-rich and acidic (Ser/Ac) domain. The 5Ј-TGTGGTGGTGAAGCTGTAGC-3Ј, ␤-actin (reverse, 40 nM), 5Ј- nuclear localization signal (NLS, f) is indicated. Numbers below indicate GACGACATGGAGA AGATCTGG-3Ј. the amino acid positions of the respective protein domains. The PIASy-␤- Gal fusion protein, schematically shown below, retains the SAP domain Two-dimensional gel electrophoresis of total cell extracts and the nuclear localization signal, but inactivates the RING and the serine- rich and acidic domains of the PIASy protein. C and D, DNA blot analysis Wild-type and Piasy-lacZ homozygous mutant Abelson MuLV-trans- of targeted ES cell clones. The targeting vector was introduced into D3 ES formed pre-B cell lines were cultured under standard conditions. Total cell Ј extracts were generated in two-dimentional gel sample buffer (7 M urea, 2 cells by electroporation. The presence of an additional 10.9 kb fragment (5 M thiourea, 2% CHAPS, 100 mM DTT, 0.2% biolyte 3-10, (Bio-Rad, external probe (C) and a 10.3 kb fragment (3Ј external probe (D) indicates Hercules, CA), 10 mM iodacetamide, 0.5 mM PMSF in the presence of homologous recombination in the ES cell clones 61, 104, 108, and 198 in standard protease inhibitors) and the total protein concentration was deter- contrast to the parental D3 ES cell line. E, DNA blot analysis of genomic mined using PlusOne 2-D Quant kit (Amersham Biosciences, Piscataway, DNA from representative litters generated by heterozygous matings using NJ). A total of 300 ␮g of protein was separated in the first dimension using the 3Ј external probe for hybridization. The 14 kb and 10.3 kb DNA frag- ReadyStrip IPG strip (Bio-Rad), pH 3–10, nonlinear, according to suppliers ments are generated from the wild-type and mutant allele, respectively. F, instructions (Bio-Rad). Focusing proceeded from 0 up to 60 kVh over 9 h Immunoblot analysis of total tissue extracts from adult testis obtained from followed by 12% SDS-PAGE in the second dimension. After fixation (5% wild-type and Piasy-lacZ homozygous mutant mice. The blot was probed with acetic acid, 5% methanol) protein spots were visualized by colloidal Coo- massie staining. For anti-SUMO1 immunoblot analysis (anti-GMP-1 a monoclonal anti-mouse PIASy Ab. Expression of the Piasy-lacZ allele yields mouse monoclonal, 1:1000; Zytomed, Berlin, Germany), two-dimensional a stable PIASy-␤-Gal fusion protein. The positions of the 60 kDa wild-type gel electrophoresis was performed as described followed by standard PIASy and the PIASy-␤-Gal fusion protein expressed by homozygous mutant blocking and immunoblotting techniques. Piasy-lacZ mice are indicated. 6192 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO construct in murine ES cells should yield cell clones in which amino acid 81 of PIASy has been fused with the N terminus of the bacterial ␤-Gal protein. This targeting strategy should allow for the visualization of the expression pattern of the Piasy gene in het- erozygous mutant mice and the inactivation of the gene in ho- mozygous mutant mice because the fusion protein lacks most of the PIASy protein domain, including the functionally important RING domain. Mutations in the RING domain have been found to abrogate the function of PIAS proteins in vitro and in transfection assays (3, 22, 33–35), and therefore we will refer to the homozy- gous Piasy-lacZ mutation also as PiasyϪ/Ϫ mutation. The linearized targeting construct was transfected into D3 ES cells and clones that had been subjected to positive and negative selection were screened for homologous recombination by digest- ing genomic DNA with EcoRV and hybridizing DNA blots with an internal probe, derived from genomic sequences at the end of intron 2 of the Piasy gene (Fig. 1A, data not shown). Ten targeted ES cell clones were identified among 108 analyzed colonies and the homologous recombination events were confirmed by hybrid- ization of XhoI/HindIII- and BamHI-digested genomic DNA with Downloaded from 5Ј and 3Ј external probes, respectively (Fig. 1, A, C, and D). Six FIGURE 2. PIASy expression during embryonic development. The ex- targeted ES cell clones were microinjected into C57BL/6 blasto- pression pattern of PIASy was determined in developing mouse embryos by X-Gal staining of whole mount E14.5 embryos (A–C), and by X-Gal cysts and chimeric males were crossed with C57BL/6 wild-type staining of 20-␮m cryostat sections of E16.5 embryos (D–F). A, Wild-type females. One chimera, derived from ES cell clone 198, transmitted control E14.5 embryo stained by X-Gal. B, Piasy-lacZ heterozygous em- the targeted allele through the germline. The genotypes of the F1 bryo stained by X-Gal. PIASy is abundantly expressed in brain, tail bud, offspring were determined by PCR (data not shown) and DNA blot limb buds, hair follicle placodes, and whisker follicles. C, X-Gal staining http://www.jimmunol.org/ analysis of BamHI-digested genomic DNA that had been hybrid- of an E14.5 Piasy-lacZ homozygous mutant embryo. No morphological ized with a 3Ј external probe (Fig. 1E). Heterozygous animals did abnormalities can be observed. D, X-Gal staining of a cryostat section of a not show any obvious differences in phenotype or fertility as com- wild-type E16.5 embryo. Background X-Gal staining was observed in the pared with wild-type littermates (data not shown). However, geno- duodenum. E, Sagittal cryosection of a Piasy-lacZ heterozygous embryo typing of 209 offspring from heterozygous intercrosses revealed a stained by X-Gal. PIASy shows a wide expression pattern in the developing significantly reduced Mendelian frequency of 14%, instead of mouse embryo. F, Sagittal cryosection of a Piasy-lacZ homozygous mutant E16.5 embryo stained by X-Gal. No morphological abnormalities can be 25%, homozygous mutant (PiasyϪ/Ϫ) mice. The Mendelian fre- observed. quency of homozygous mutant PiasyϪ/Ϫ embryos up to E18.5 was by guest on October 2, 2021 normal, suggesting a decreased perinatal viability. The surviving PIASy-deficient mice showed no obvious phenotype and were fertile. ␤-Gal staining was detected, except the duodenum, indicating the To confirm that the targeted Piasy allele does not produce func- observed expression in heterozygous mice is specific (Fig. 2D). tional PIASy protein (Fig. 1B), we prepared total protein extracts These results correspond to previous in situ hybridization studies from testis of wild-type and homozygous mutant (PiasyϪ/Ϫ) mice of E8.5 up to E16.5 mouse embryos, which detected Piasy tran- and assayed the extracts for the presence of PIASy protein by scripts in the limbs, the neuroepithelium, and hair follicles (55). immunoblot analysis. Using a rat anti-PIASy mAb directed against Taken together, these studies suggest that PIASy may play a mod- amino acids 1 to 97 of the mouse PIASy protein, we failed to ulatory role during the development of multiple organs. Ϫ Ϫ detect wild-type PIASy protein in the mutant tissue extract, al- Morphological analysis of sections of Piasy / mice at E14.5 though the slower-migrating PIASy-␤-Gal fusion protein was and E16.5 did not reveal any obvious abnormality (Fig. 2, C and readily detectable (Fig. 1, B and F). F). In addition, no altered expression pattern or mislocalization of the PIASy-␤-Gal fusion protein is detected in Piasy-lacZ homozy- Analysis of Piasy expression during embryonic development and gous mutant mice (Fig. 2F). Although adult PiasyϪ/Ϫ mice also morphology of PIASy-deficient mice show no obvious phenotype, the basis for the reduced viability of With the aim of visualizing individual cells and tissues that express these mice remains to be established. the Piasy gene in the embryo, we performed whole mount ␤-Gal staining of heterozygous Piasy-lacZ embryos at E12.5 and E14.5 PIASy expression in hemopoietic cells and analysis of cell (Fig. 2B and data not shown). Abundant expression of PIASy-␤- populations in PIASy-deficient mice Gal was detected in multiple tissues, including brain, skin, limb To address the question of whether PIASy is specifically expressed buds, tail bud, whisker follicles, and pelage hair follicle primordia in distinct hemopoietic cell populations, we incubated cells of lym- (Fig. 2B). In sagittal cryosections of heterozygous Piasy-lacZ em- phoid organs with fluoresceine-digalactoside, a fluorogenic sub- bryos at E16.5, an almost ubiquitous expression pattern could be strate for ␤-Gal to detect Lac-Z-positive cells by flow cytometry. observed with stronger expression in the neopallial cortex and the Using this approach, we found that PIASy is abundantly expressed ventricular zone of the brain, the midbrain, the developing cere- in developing and mature T cells of the thymus (Fig. 3A). Histo- bellum, throughout the spinal cord and in the olfactory epithelium gram overlays of cells gated for PIASy-␤-Gal expression indicated (Fig. 2E). Strong PIASy expression was also detected in the epi- that 98–99% of CD4ϩCD8ϩ double positive cells, CD4ϩ single dermis, the dorsal surface of the tongue, in the trapezius muscle positive and CD8ϩ single positive cells express PIASy at abundant and the cartilage primordium of the ribs and bones, the thyroid levels (Fig. 3A). Analysis of the T cell compartments in thymus cartilage, tracheas, and lung (Fig. 2E). In wild-type embryos, no and spleen of wild-type mice and Piasy-lacZ homozygous mutant The Journal of Immunology 6193 mice by flow cytometry indicated that the numbers of CD4ϩCD8ϩ mined the levels of Axin2 mRNA in MEF cell lines that have been double positive cells, as well as the numbers of CD4ϩ and CD8ϩ treated with LiCl, which inhibits glycogen-synthase-kinase-3␤ and single positive cells, are equivalent in both wild-type and mutant acts as a surrogate Wnt signal (61). For this experiment, we used mice (Fig. 3, B and C). two wild-type and four mutant mice to derive primary MEFs, and B220ϩCD43ϩ pre-B cells of the bone marrow and mature B we performed the expression analysis with duplicate samples in cells of the spleen (B220ϩIgMϩ) were found to express high levels three independent experiments. Determination of basal Axin2 lev- of PIASy-␤-Gal (Fig. 3, D and F). However, the analysis of the B els in untreated MEFs by quantitative real-time PCR analysis did cell compartments in bone marrow and spleen of homozygous mu- not reveal any significant differences between wild-type and Piasy- tant mice showed a normal B220/CD43 profile and B220/IgM pro- lacZ homozygous mutant cells (Fig. 5A). Stimulation of the MEF file, respectively (Fig. 3, E and G). cells derived with LiCl resulted in quite variable induction effi- Finally, PIASy-␤-Gal expression was detected at lower levels in ciencies, ranging from 5.6-fold (PiasyϪ/Ϫ MEF 17) up to 24.2-fold myeloid cells of bone marrow, using the markers Mac1 and GR1 (PiasyϪ/Ϫ MEF 12) (Fig. 5A). The induction efficiencies of the (Fig. 3H). Analysis of the myeloid compartment in the bone mar- wild-type MEF cell lines ranged between 7.8-fold (wild-type MEF row of PiasyϪ/Ϫ mice showed modest but reproducible 20% in- 5) and 13.1-fold (wild-type MEF no. 18). Therefore, the Wnt re- crease in the numbers of Mac1ϩGR1ϩ double positive cells (Fig. sponse of Axin2 in PIASy-deficient MEFs is comparable to that in 3I). Thus, PIASy is abundantly expressed in multiple cell types of wild-type MEFs. the lymphoid and myeloid lineages. However, PIASy is not es- To further assess the Wnt responsiveness of wild-type and sential for normal B and T cell development and has a modest PiasyϪ/Ϫ MEFs, we transfected transiently these cells with a LEF1 effect on myeloid cell differentiation. reporter construct, in which multimerized binding sites for LEF1/ TCF proteins are linked to the minimal c-fos promoter and lucif- Downloaded from Cytokine signaling in Piasy-lacZ homozygous mutant mice erase gene (LEF7-fos-luciferase). Previously, we found that PIASy IFN-␣ and IFN-␥ signal to the nucleus by activating the latent represses the LEF1- and ␤-catenin-dependent activation of this cytoplasmic transcription factor STAT1, which is translocated to reporter construct in transiently transfected 293 cells (3). In a first the nucleus and activates IFN-responsive target genes (reviewed in experiment, we compared the expression levels of the transfected Refs. 6 and 7). PIASy was shown to act as a transcriptional core- LEF1 reporter in wild-type with PIASy-deficient MEF cells in the pressor of STAT1 and the association of these proteins was found absence or presence of the surrogate Wnt signal, LiCl, but without http://www.jimmunol.org/ to be augmented by IFN signals (27). This observation has been the addition of exogenous LEF1 or ␤-catenin expression constructs interpreted to suggest that PIASy plays a role in the attenuation of (Fig. 5B). The basal levels of reporter activity were similar in the IFN response. STAT1 is indispensable for the IFN pathways NaCl-treated wild-type and mutant MEF cells (Fig. 5B). LiCl treat- (56, 57) and therefore, we examined the ability of IFN-␣ and ment of these cells induced a 2- to 3-fold increase in reporter gene IFN-␥ to induce target gene transcription in splenocytes and bone expression in both wild-type and mutant MEFs, suggesting that the marrow-derived macrophages from wild-type or Piasy-lacZ ho- loss of functional PIASy had no effect on basal or Wnt-induced re- mozygous mutant mice. Using quantitative real-time PCR analysis porter gene activity. Because the effects of LiCl in MEFs were lim- to detect transcripts of Irf1 gene, a direct target of STAT1 (58, 59), ited, possibly due to the presence of low levels of endogenous LEF1/ by guest on October 2, 2021 we found that Irf1 gene expression was induced 5- to 9-fold in TCF proteins, we compared the activity of the LEF7-fos-luciferase IFN-␣-treated wild-type splenocytes (Fig. 4A) and 7- to 17-fold in reporter in wild-type and Piasy-lacZ homozygous mutant MEFs in the bone marrow-derived wild-type macrophages (Fig. 4B). For these presence of exogenous LEF1 and ␤-catenin. Cotransfection of LEF1 experiments, we analyzed duplicate samples of two mice to assess and ␤-catenin expression plasmids into wild-type MEFs resulted in a the variabilities of the nuclear responses to cytokine signaling. robust 11.2-fold activation of the LEF7-fos-luciferase reporter (Fig. IFN-␥-stimulation resulted in a 14- to 16-fold augmentation of Irf1 5C, wt MEF 18). However, in PiasyϪ/Ϫ MEFs, the transcriptional expression in wild-type splenocytes (Fig. 4A, PIASyϩ/ϩ) and ϳ26- activation of the reporter construct was reduced almost 3-fold (Fig. fold stimulation in wild-type macrophages (Fig. 4B, PIASyϩ/ϩ). 5C, MEF 11), whereas no effect was observed in cells that have been In splenocytes and macrophages from PIASy-deficient mice, the transfected with only LEF1 or ␤-catenin expression plasmids. This basal and IFN-␣-induced levels of Irf1 mRNA expression were result suggests that PIASy may modulate the Wnt responsiveness of both similar to those of wild-type mice (Fig. 4), suggesting that the genes in vivo. response to IFN-␣ is normal in the absence of PIASy. However, The modest effects of the deficiency of PIASy could be, in prin- the response of the mutant splenocytes and macrophages to IFN-␥ ciple, accounted for by a redundancy with another member of the was reproducibly reduced by a factor of 1.5 and 2, respectively PIAS family and/or an up-regulation of the expression of another (Fig. 4). In particular, the stimulation of wild-type macrophages by PIAS gene. Therefore, we examined the expression of the PIAS IFN-␥ resulted in an ϳ26-fold induction of Irf1, whereas only a 9- genes in wild-type and PiasyϪ/Ϫ MEFs. To this end, we performed to 16-fold induction was observed in the PIASy-deficient cells a quantitative real-time PCR analysis to detect transcripts of the (Fig. 4B). Pias1, Pias3, and Piasx genes. The levels of expression of these Pias genes were found to be similar in wild-type and PIASy-de- Wnt signaling in Piasy-lacZ homozygous mutant mice ficient MEFs, suggesting that the absence of PIASy does not result LEF1/TCF proteins are nuclear mediators of Wnt signals that ac- in a compensatory up-regulation of the expression of another Pias tivate transcription of Wnt-responsive target genes in conjunction gene (data not shown). ␤ with -catenin (reviewed in Ref. 10). LEF1 and TCF4 were found Ϫ/Ϫ to interact specifically with PIASy, whereby the activity of LEF1 Two-dimensional gel electrophoresis of wild-type and Piasy is repressed and the activity of TCF4 is enhanced (3, 4). Biochem- cell extracts ical and genetic analyses identified Axin2, which acts as a negative To investigate whether PIASy modulates the expression of Wnt or regulator of soluble ␤-catenin, as a target of Wnt signaling, thus STAT target genes and to assess whether a deficiency of PIASy establishing a negative feedback loop for Wnt signaling (60). To alters the SUMO modification of proteins, we adopted a protein examine the Wnt responsiveness of the endogenous Axin2 gene in expression profiling approach. Total cell extracts of wild-type and wild-type and Piasy-lacZ homozygous mutant mice, we deter- PiasyϪ/Ϫ Abelson-MuLV-transformed pre-B cells were separated 6194 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. PIASy expression analysis in hemopoietic cells and phenotypic characterization of the hemopoietic system of Piasy-lacZ homozygous mutant mice. The expression of PIASy was determined in heterozygous Piasy-lacZ mice using fluorescein-digalactoside (FDG), a fluorogenic substrate for ␤-galactosidase (␤-Gal). A, PIASy expression in the thymus. Thymocytes from heterozygous Piasy-lacZ mice were stained for the expression of CD4, CD8, and PIASy-␤-Gal. The representative CD4/CD8 profile indicating the selected gates and quadrant statistics is shown on the left. Right, overlay histograms of PIASy-␤-Gal expression in CD4ϩ/CD8ϩ, CD4ϩ, and CD8ϩ cells indicate that PIASy is expressed in all three compartments. B and C, CD4/CD8 profile of thymic (B) and splenic (C) T cells from wild-type (ϩ/ϩ) and Piasy-lacZ homozygous mutant mice. The T cell compartment in Piasy-lacZ homozygous mutant mice is normal. D and F, PIASy expression analysis in the B cell compartment of bone marrow (D) and spleen (F). Total bone marrow cells (D) and splenocytes (F) from heterozygous Piasy-lacZ mice were stained for the expression of B220, CD43, IgM, and PIASy-␤-Gal. D, The B220/CD43 profile with the selected gate for the PIASy expression analysis in bone marrow (6.1% of living cells) is shown on the left. Right, the overlay histogram of PIASy-␤-Gal expression in the B220ϩCD43ϩ double positive cell population indicates decent PIASy expression in developing B cells. F, Overlay histograms of PIASy-␤-Gal expression of B220ϩ and IgMϩ single positive B cells. The percentage of cells gated for the PIASy-␤-Gal expression analysis is indicated in brackets. PIASy is expressed in splenic B cells. E and G, Analysis of the B cell compartment of bone marrow (E)(Figure legend continues) The Journal of Immunology 6195 in two dimensions by isoelectric focusing followed by SDS- PAGE. Protein spots were visualized by colloidal Coomassie stain- ing and the gels were scanned for PDQUEST (Bio-Rad) software analysis (Fig. 6, A and B). We prepared pre-B cell extracts from two wild-type and two PIASy-deficient mice and assayed the ex- tracts in duplicates by two-dimensional electrophoresis (Fig. 6, A and B). The comparison of the pattern of Coomassie-stained pro- tein spots in wild-type and mutant cell extracts by the PDQUEST software (Bio-Rad) showed no differential expression pattern of proteins or shifts in protein migration due to potential secondary modifications of proteins comparing total cell extracts from wild- type (Fig. 6A) and Piasy-lacZ homozygous mutant Abelson MuLV-transformed pre-B cells (Fig. 6B). To identify potential protein substrates for SUMO modification by PIASy, total protein extracts of wild-type and Piasy-lacZ ho- mozygous mutant Abelson MuLV-transformed pre-B cells were separated by two-dimensional gel electrophoresis and transferred to nitrocellulose membranes for immunoblot analysis using a mouse monoclonal anti-SUMO1 Ab (anti-GMP-1; Zytomed). No significant difference in the pattern of immunoreactive spots could Downloaded from be detected comparing the anti-SUMO1 immunoblots from wild- type and Piasy-lacZ homozygous mutant Abelson MuLV-trans- formed pre-B cells (Fig. 6, C and D).

Discussion To date, the functional analysis of mammalian PIAS proteins, http://www.jimmunol.org/ which identified these proteins both as regulators of nuclear me- diators of various signaling pathways and as SUMO E3 ligases, involved transient transfections and overexpression of both PIAS FIGURE 4. Transcriptional responses to IFN-␣ and IFN-␥ in spleno- and partner proteins in cell lines. With the aim of determining the cytes and macrophages of Piasy-lacZ homozygous mutant mice. A, Spleno- biological role of PIAS proteins in a more physiological context, cytes derived from wild-type and Piasy-lacZ homozygous mutant mice we undertook the genetic inactivation of a particular member of were treated with 500 U/ml murine IFN-␣ and 30 ng/ml murine IFN-␥ for the PIAS family, PIASy, and examined the effects of the mutation 2 h, respectively. Total RNA was extracted and the level of the STAT1 target gene Irf1 was determined by quantitative real-time PCR. Basal Irf1 in primary cells. This analysis revealed a modest reduction of the by guest on October 2, 2021 transcriptional response to IFN-␥. In addition, we observed a re- expression (e) is shown. IFN-␣-stimulated (f) and IFN-␥-stimulated ( ) duced viability of the homozygous mutant mice and found that the Irf1 expression are indicated. The basal expression of C57BL/6 is set to activation potential of a Wnt-responsive reporter by LEF1 and one. The basal Irf1 expression levels of the other animals are presented relative to C57BL/6 and are very similar. The fold inductions were calcu- ␤-catenin is decreased. Together, these data suggest that PIASy ␥ lated relative to the corresponding basal level. B, Bone marrow macro- may play a regulatory role in signaling by IFN- and Wnt proteins phages derived from wild-type and Piasy-lacZ homozygous mutant mice in vivo. were expanded as outlined in Materials and Methods. Macrophage cultures The subtle effects of the Piasy-lacZ mutation raise the question were treated with 500 U/ml murine IFN-␣ and 30 ng/ml murine IFN-␥ for of whether the insertion of the lacZ gene into the second exon of 2 h, respectively. Total RNA was extracted and real-time PCR analysis was Piasy generates a null allele or whether homozygous Piasy-lacZ performed to determine the transcriptional response of the STAT1 target mice retain a residual activity of PIASy protein. The immunoblot gene Irf1. Isolated splenocytes and macrophages from Piasy-lacZ homozy- analysis of testis tissue extracts from mutant mice indicates that gous mutant mice respond to IFN-␣ to a similar extent as cells from wild- ␥ neither wild-type PIASy nor smaller forms of PIASy protein can type mice. However, the transcriptional response to IFN- is reduced in be detected. This observation rules out the principle possibility that PIASy-deficient mice by a factor of two to three. exon skipping generates a PIASy protein, lacking only the exon with the lacZ insertion. Although the N-terminal SAP domain is retained in the PIASy-␤-Gal fusion protein, this protein lacks the Several lines of evidence suggest that other PIAS proteins may central RING domain, which contributes to the interaction with compensate, at least in part, for the deficiency of PIASy in the partner proteins and is essential for the functional activity of mouse. First, PIAS proteins share a similar domain structure and PIASy. Mutations in the RING domain of PIASy and other PIAS show overlapping specificities in the association and sumoylation proteins have been shown to abrogate the ability of these proteins of partner proteins. For example, transient overexpression of to modulate the activity of partner proteins and act as SUMO E3 PIASy and PIAS1 both result in the repression of a STAT1-re- ligases (3, 22, 34, 35). sponsive reporter construct in transfection experiments (2, 27).

and spleen (G) in wild-type and Piasy-lacZ homozygous mutant mice using the cell surface markers B220, CD43 for bone marrow (E), and B220, IgM for spleen (G). No abnormalities can be observed in the B cell compartment of Piasy-lacZ homozygous mutant mice. H, PIASy expression in the myeloid lineage. Total bone marrow cells from heterozygous Piasy-lacZ mice were stained for the expression of Mac1, GR1, and PIASy-␤-Gal. The Mac1/GR1 profile with the selected gate for the PIASy expression analysis (42.4% of the living cell population) is shown on the left. Right, the overlay histogram of PIASy-␤-Gal expression in Mac1ϩGR1ϩ double positive cells indicates that PIASy is expressed. I, Mac1 and GR1 expression analysis in bone marrow of wild-type and Piasy-lacZ homozygous mutant mice. No major abnormality can be observed in myeloid cells of Piasy-lacZ homozygous mutant mice. 6196 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO

fection assays. PIASx␣, but not PIAS3, antagonizes the IL-12- dependent activation of a reporter gene by STAT4 (5). Likewise, the repression and sumoylation of the nuclear matrix protein SATB2 is mediated specifically by PIAS1, but not by PIASx and PIAS3 (62). Second, PIAS proteins are widely expressed and show overlapping pattern of expression in the developing mouse (55). Our analysis of heterozygous and homozygous Piasy-lacZ em- bryos shows an almost ubiquitous expression at a low level but a more specific expression in multiple tissues. Third, a redundancy of PIAS proteins has been found in yeast, in which two PIAS- related proteins, termed Siz1 and Siz2, act redundantly in promot- ing SUMO modification of septins (14). Both Siz1⌬ and Siz2⌬ strains show no obvious phenotypes, but the double mutation re-

sulted in poor cell growth and accumulation in G2/M of the cell cycle. Moreover, the overall sumoylation of proteins was found to be diminished in Siz1⌬ cells but was virtually abrogated in Siz1⌬Siz2⌬ cells (14). One obvious effect of the Piasy-lacZ mutation was the reduced viability of newborn mice. In contrast to the normal Mendelian frequency of homozygous mutant mice before birth, we observed Downloaded from only 14% homozygous mutants at weaning age. As we could not detect any obvious abnormalities, the basis for the reduction of the viability is unknown. The reduced viability may also reflect some heterogeneity in the genetic background of the mice, which were generated in a 129 strain but were backcrossed to a C57BL/6 strain. Strain-specific variations in the penetrance of mutations http://www.jimmunol.org/ have been observed (63). Although PIASy is expressed in multiple hemopoietic cell lin- eages, the only effect of the Piasy-lacZ mutation that we observed was a 25% increase in the number of Mac1- and Gr1-positive myeloid cells in the bone marrow. This modest increase in myeloid cells is reproducible in multiple mice and is consistent with the finding that the expression of Pias3 is lost in leukemic cells in which STAT3 is persistently activated (64, 65). Although PIASy by guest on October 2, 2021 has not been implicated in the regulation of STAT3, it is possible that PIASy and PIAS3 act redundantly in myeloid cells to modu- late cytokine signaling. In macrophages of the bone marrow and to a lesser extent in FIGURE 5. Analysis of Wnt responsiveness in wild-type vs Piasy-lacZ splenocytes, we detected a reproducible 2- to 3-fold decrease in the homozygous mutant MEFs. A, Axin2 mRNA levels were determined by IFN-␥ responsiveness of the endogenous Irf1 gene, a member of real-time PCR analysis of wild-type and Piasy-lacZ homozygous mutant the IRF family of transcription factors, that is a direct target gene MEFs after stimulation with 30 mM LiCl (as a surrogate Wnt signal, (Ref. of IFN signaling (58, 59). IRF1 is involved in the regulation of the 61) and 30 mM NaCl (control) for 8 h. Axin2 mRNA levels were normal- expression of both IFN-stimulated genes and IFN genes them- ␤ ized to -actin and the fold-change was calculated by setting the normal- selves. The induction of Irf1 transcription by IFN-␥ in wild-type ized relative Axin2 level of the unstimulated (30 mM NaCl treatment) macrophages appears to be governed specifically by STAT1 be- wild-type control MEF cell line no. 5 to one. Basal Axin2 levels (unstimu- ␥ lated, 30 mM NaCl, e) do not differ between wild-type and mutant MEFs. cause no Irf1 induction is observed in IFN- -treated macrophages Ϫ/Ϫ ␥ Numbers above the bars indicate the fold-change of the Axin2 mRNA of Stat1 mice (56, 57). The decrease in the IFN- response of levels comparing each unstimulated (30 mM NaCl, e) with stimulated (30 Irf1 is surprising, given the proposed function of PIAS proteins in mM LiCl, f) MEF cell line of the indicated genotype. The data were the down-regulation of the activities of STAT proteins. Several derived from three independent experiments performed in duplicates. B, possibilities could account for this difference. First, in our analysis ␮ Wnt-responsiveness of the LEF7-fos-luciferase reporter (1 g) was exam- we examine the effects of the PIASy deficiency in the context of a ined in transient transfection assays of wild-type vs mutant MEFs by treat- natural gene. In contrast, the effects of PIASy on STAT1 activity ment of transfected cell pools with 30 mM LiCl as a surrogate Wnt stim- were determined with synthetic reporter constructs containing ulus and 30 mM NaCl as control, respectively. Numbers given indicate the multimerized STAT1 binding sites, and in conjunction with over- fold-changes in reporter gene activity in response to LiCl relative to the expressed PIASy protein (27). The context of transcription factor control NaCl treatment, which is set to one. C, LEF1-dependent reporter gene activity was examined by transient transfection assays of wild-type binding sites has been shown to be important for the proper reg- ␮ ulation of the IFN-␤ gene by IRF, NF-␬B, ATF2, and HMG-I/Y, and mutant MEFs using 1 gofLEF7-fos-luciferase reporter in the absence or presence of 50 ng of Lef1 and 0.5 ␮gof␤-catenin. Data shown are which act in concert to mediate a proper transcriptional response duplicates of a representative transfection. (66, 67). For example, replacement of the ATF2-c-jun site with an AP-1 site in the IFN-␤ promoter, or reversal of the orientation of the asymmetric ATF2-c-jun site impairs its virus induction (68). Moreover, PIAS1 and PIASx proteins both act as SUMO E3 li- Therefore, changes in the context of transcription factor binding gases and corepressors of AR and p53 (12, 22, 34, 35). However, sites can result in different regulatory contributions of proteins. some specificity of PIAS proteins can be demonstrated in trans- Second, we examined the effects of PIASy in primary cells and The Journal of Immunology 6197 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. Two-dimensional gel electrophoresis analysis of wild-type and Piasy-lacZ homozygous mutant pre-B cell lines. Total protein extracts were generated from MuLV transformed pre-B cell lines derived from wild-type (ϩ/ϩ) and Piasy-lacZ homozygous mutant embryos. A total of 300 ␮g of total protein were separated by isoelectric focusing in the first dimension using a nonlinear gradient of pH 3–10, followed by 12% PAGE in the second dimension. A and B, Colloidal Coomassie staining of two-dimensional gels performed from wild-type (A) and Piasy-lacZ homozygous mutant (B) total protein extracts. No major differences in the overall staining pattern of wild-type and mutant extracts could be observed. C and D, Anti-SUMO1 immunoblot analysis of two-dimensional gels performed from wild-type (C) and Piasy-lacZ homozygous mutant (D) total protein extracts. No significant differences in the immunoreactive patterns of wild-type and Piasy-lacZ homozygous mutant protein extracts could be observed. without the overexpression of proteins, which occurs typically in these other cytokines, whereas both IFN-␣- and IFN-␥-signaling transfection assays and may squelch a regulatory response by ti- was impaired, indicating that STAT1 is a transcription factor ded- trating regulatory cofactors. Although the relatively modest effect icated to IFN signaling (56, 57, 75). The basis for the differential of the PIASy deficiency on Irf1 expression may reflect a redun- effect of PIASy on signaling by IFN-␣ and IFN-␥ is unclear but it dancy of PIASy with other PIAS proteins, such as PIAS1, we may be related to the cross-talk between these signaling pathways cannot rule out the possibility that the contribution of PIAS pro- (6). teins to the regulation of signaling by STAT proteins is limited. Based on the association of LEF1/TCF proteins with PIASy in The overexpression of PIASx␣ augments the activation of a syn- vitro, we also anticipated an alteration in the expression of a Wnt- thetic reporter construct by STAT1 in IFN-␥-treated U3a cells, responsive reporter construct by LEF1 and ␤-catenin in PIASy- which lack endogenous STAT1 only 2-fold (36). deficient MEFs. We have previously shown that PIASy represses In contrast to the modest reduction of the IFN-␥ response in the activity of LEF1 in transiently transfected HeLa or 293 cells PIASy-deficient macrophages and splenocytes, no changes could (3). In addition, PIASy was found to augment the activation of a be observed in the IFN-␣ response. Both IFNs use STAT1 as a LEF1/TCF reporter by TCF4 and ␤-catenin (4), which is consistent nuclear mediator but the responses differ in the specificity of tran- with our observation that the activation potential of LEF1 in scription factor activation. IFN-␥ activates STAT1 as a dedicated PIASy-deficient MEFs is reduced relative to that in wild-type transcription factor, whereas IFN-␣ activates both STAT1 and MEFs. In our previous transfection experiments, in which we ob- STAT2 (69, 70). In addition, other signaling molecules have been served repression of the LEF1 activity by PIASy, we have used found to activate STAT1 in vitro. These have been shown to in- 293 and HeLa cells and noted that the repression was not observed clude epidermal growth factor, M-CSF, IL-6, IL-10, and others in fibroblastic cells, suggesting that the regulation of transcription (71–74). STAT1-deficient mice show a normal responsiveness to factor activity by PIASy is cell type-specific (3). In contrast to the 6198 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO changes of LEF1- and ␤-catenin- mediated activation of a reporter 13. Ro¨del, B., K. Tavassoli, H. Karsunky, T. Schmidt, M. Bachmann, F. Schaper, construct in PIASy-deficient MEFs, we did not detect any signif- P. Heinrich, K. Shuai, H. P. Elsa¨sser, and T. Mo¨ro¨y. 2000. The zinc finger protein Gfi-1 can enhance STAT3 signaling by interacting with the STAT3 inhibitor icant change in the Wnt-response of the endogenous Axin2 gene, a PIAS3. EMBO J. 19:5845. known Wnt target gene (60). In MEFs, the amount of LEF/TCF 14. Johnson, E. S., and A. A. Gupta. 2001. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106:735. proteins is possibly too low to elicit a strong Wnt response and 15. Kahyo, T., T. Nishida, and H. Yasuda. 2001. Involvement of PIAS1 in the PIASy may have an effect only under conditions of strong Wnt sumoylation of tumor suppressor p53. Mol. Cell 8:713. signals. 16. Takahashi, Y., T. Kahyo, A. Toh-e, H. Yasuda, and Y. Kikuchi. 2001. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as A common hallmark of PIAS proteins is their ability to augment an adaptor between conjugating enzyme and substrates. J. Biol. Chem. the modification of proteins with SUMO (38). Therefore, we ex- 276:48973. amined whether the sumoylation pattern of proteins is altered in 17. Zentner, M. D., H. H. Lin, H.-T. Deng, K.-J. Kim, H.-M. Shih, and D. K. Ann. 2001. Requirement for high mobility group protein HMGI-C interaction with PIASy-deficient cells. In transformed pre-B cells from mutant STAT3 inhibitor PIAS3 in repression of ␣-subunit of epithelial Naϩ channel mice, we did not detect a significant change in the pattern of pro- (␣-ENaC) transcription by Ras activation in salivary epithelial cells. J. Biol. tein expression relative to wild-type mice. Notably, the sumoyla- Chem. 276:29805. 18. Kotaja, N., M. Vihinen, J. J. Palvimo, and O. A. Ja¨nne. 2002. Androgen receptor- tion of proteins by PIAS proteins does not necessarily correlate interacting protein 3 and other PIAS proteins cooperate with glucocorticoid re- with changes in their activity. For example, PIAS1 and PIASx␣ ceptor-interacting protein 1 in steroid receptor-dependent signaling. J. Biol. Chem. 277:17781. both modulate the activity of STAT1 in transfection assays (36, 19. Nakagawa, K., and H. Yokosawa. 2002. PIAS3 induces SUMO-1 modification 40). However, the sumoylation of STAT1 by PIASx␣ does not and transcriptional repression of IRF-1. FEBS Lett. 530:204. correlate with changes in transcriptional activation as a mutation of 20. Ross, S., J. L. Best, L. I. Zon, and G. Gill. 2002. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Mol. the SUMO acceptor site at lysine 703 of STAT1 has no effect on Cell. 10:831. Downloaded from STAT1 activity (36). Likewise, mutations of two SUMO acceptor 21. Sapetschnig, A., G. Rischitor, H. Braun, A. Doll, M. Schergaut, F. Melchior, and sites of LEF1 have no effect on the activity of LEF1, although the G. Suske, G. 2002. Transcription factor Sp3 is silenced through SUMO modifi- cation by PIAS1. EMBO J. 21:5206. association with PIASy and the presence of an intact RING do- 22. Schmidt, D., and S. Mu¨ller. 2002. Members of the PIAS family act as SUMO main is required for the repression of LEF1 in 293 cells (3). In ligases for c-Jun and p53 and repress p53 activity. Proc. Natl. Acad. Sci. USA contrast, sumoylation of some proteins, such as AR and SATB2 99:2872. 23. Lee, P. S. W., C. Chang, D. Liu, and R. Derynck. 2003. Sumoylation of Smad4, alters their activities (35, 62). Thus, PIAS proteins may use mul- the common Smad mediator of transforming growth factor-␤ family signaling. tiple mechanisms to modulate the activities of partner proteins. J. Biol. Chem. 278:27853. http://www.jimmunol.org/ 24. Long, J., I. Matsuura, D. He, G. Wang, K. Shuai, and F. Liu. 2003. Repression In conclusion, the analysis of the PIASy-deficient mice has of Smad transcriptional activity by PIASy, and inhibitor of activated STAT. Proc. revealed a functional role for PIASy in the viability of mice and Natl. Acad. Sci. USA 100:9791. IFN-␥ signaling, although the effects are modest. Therefore, it 25. Tussie´-Luna, M. I., D. Bayarsaihan, E. Seto, F. H. Ruddle, and A. L. Roy. 2002. Physical and functional interactions of histone deacetylase 3 with TFII-I family will be of interest to examine the effects of mutations of mul- proteins and PIASx␤. Proc. Natl. Acad. Sci. USA 99:12807. tiple Pias genes in the mouse to determine the role of PIAS 26. Gross, M., B. Liu, J. Tan, F. S. French, M. Carey, and K. Shuai. 2001. Distinct proteins in sumoylation and regulation of transcription factor effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells. Oncogene 20:3880. activity. While this study was under review, an independent 27. 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