Tip60 Is a Co-repressor for STAT3*

Hui XiaoDagger , Jin ChungDagger , Hung-Ying Kao§, and Yu-Chung YangDagger ||

From the Dagger  Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and the § Department of Biochemistry and Cancer Center, Case Western Reserve University, School of Medicine Cleveland, Ohio 44106

Received for publication, October 22, 2002, and in revised form, January 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tip60 (Tat-interactive protein, 60 kDa), a cellular protein with intrinsic histone acetyltransferase activity, is involved in DNA damage repair and apoptosis. Recent studies have suggested that Tip60 acts either as a co-activator or a co-repressor to modulate transcription. In this study, we demonstrate that Tip60 represses reporter gene expression when it is fused to the Gal4 DNA binding domain. We also show that Tip60 associates with histone deacetylase 7 (HDAC7) through its N-terminal zinc finger-containing region and that HDAC7 activity is required for the repressive effect of Tip60. Because endogenous Tip60 interacts with STAT3, we hypothesized that Tip60 might complex with STAT3 and HDAC7 and modulate STAT3-mediated trans-activation. Consistent with this hypothesis, the overexpression of Tip60 represses STAT3-driven reporter gene expression, which can be further potentiated by the co-transfection of HDAC7. Furthermore, interleukin-9-induced c-myc expression, which depends on STAT3 activity, is abrogated by exogenous expression of Tip60. This is the first demonstration of which Tip60 represses STAT3 activity in part through the recruitment of HDAC7.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tip601 (Tat-interactive protein, 60 kDa) is a member of the MYST family of proteins, which are highly conserved from yeast to human and play diverse physiological functions (1). Several members among this family, such as SAS3 (Something About Silencing), Esa1 (Essential sas2-related acetyltransferase), and Tip60, possess intrinsic histone acetyltransferase (HAT) activity (2-4), suggesting their potential roles in chromatin remodeling and gene regulation. Tip60 is expressed in a variety of tissues and cell lines, and its homologues have been identified in chicken, mouse, and human (5-7). Tip60 is mainly localized in the nucleus; however, cytoplasmic and perinuclear localization has been reported previously (4, 8-11). Tip60 forms stable nuclear complexes, which possess ATPase and DNA helicase activities, that promote histone acetylation in nucleosomes (12). It associates with transcriptional activators, such as HIV-1 Tat, type I nuclear hormone receptors, and APP (beta -Amyloid Precursor Protein), to activate gene expression (7, 13, 14). Tip60 has also been implicated in the negative regulation of gene expression through binding to CREB or the transcriptional repressor ZEB (Zinc Finger E Box-binding Protein) (15, 16). Interestingly, Tip60 interacts with membrane receptors for IL-9 and endothelin (10, 11), suggesting its involvement in signal transduction in response to extracellular stimuli. Cytosolic phospholipase A2-interacting protein, a differentially spliced form of Tip60, interacts with cytosolic phospholipase A2 to enhance cytosolic phospholipase A2-mediated cell death and prostaglandin E2 production (9).

Eucaryotic genomic DNA is packaged with histones into nucleosomes, which are the primary structural units of chromatin. The packaging of DNA into chromatin inhibits transcription in part by hindering the binding of transcription factors and basal transcriptional machinery. Many transcriptional co-activators possess intrinsic HAT activity that provides a link between histone acetylation and transcriptional regulation (17). The identification and characterization of histone deacetylases provide support for the view that reversible acetylation of histones plays an important role in gene regulation (18). Mammalian class I HDACs composed of HDAC1-3, HDAC8, and HDAC11 are highly homologous to the yeast gene, Rpd3 (19, 20). This class of deacetylases is present in multisubunit complexes such as Sin3/HDAC (21) and NuRD/Mi2/NRD (22) as well as SMRT- and nuclear receptor co-repressor-containing complexes (23, 24). Class II HDACs, including HDAC4-7 and HDAC9/10, are homologous to the yeast gene, Hda1 (20). Class II HDACs have been implicated in gene regulation through association with co-repressors such as SMRT (silencing mediator for retinoic acid and thyroid hormone receptor), nuclear receptor co-repressor, BCoR, and transcription factors of the MEF2 and POK family members (25-29).

Many cytokines, hormones, and growth factors utilize STAT signaling pathways to induce diverse biological responses including development, cell proliferation, differentiation, and survival (30). STAT3 is a STAT family member involved in normal cellular responses and oncogenesis (31, 32). Through the induction of genes involved in cell cycle progression, apoptosis, and cell motility, STAT3 plays a crucial role in mediating the physiological effects of many cytokines. STAT3 null mice die early in embryogenesis prior to gastrulation, possibly because of deficient leukemia inhibitory factor signaling (33). Tissue-specific STAT3 knock-out studies suggest a role for STAT3 in IL-10-mediated anti-inflammatory responses, growth factor-mediated migration of epidermal cells, and IL-6-mediated effects on T cells (31). We and others (34, 35) demonstrated that STAT3 acts in synergy with insulin receptor substrate-1/2 signaling to induce the proliferative and anti-apoptotic effects of IL-9. In addition, STAT3 has been implicated in tumor initiation and progression. Constitutive activation of STAT3 is observed in a wide variety of human cancers, and blockade of constitutive STAT3 signaling results in growth inhibition and apoptosis of tumor cells (36). Furthermore, a constitutively active mutant form of STAT3 induces oncogenic transformation of NIH 3T3 cells (37).

Considering the importance of STAT3 in transmitting the biological effects of extracellular stimuli and the potential relevance of STAT3 to oncogenesis, it is not surprising that many interacting partners may regulate STAT3 activity. Co-activators CBP/p300 and NCoA/SRC1a, which possess intrinsic HAT activity, interact with STAT3 and enhance its transcriptional activity (38, 39). Transcriptional activators such as c-Jun, Sp1, and GR associate with and act in synergy with STAT3 to activate gene expression (40). Tyrosine phosphatases and suppressor of cytokine signaling #3 negatively regulate STAT3 activation (41). PIAS3 (protein inhibitor of activated STAT3), a protein that specifically binds to activated STAT3, inhibits STAT3 transcriptional activity by reducing DNA binding (42). Therefore, studies of STAT3 regulatory mechanisms will not only further our understanding of how STAT3 exerts its physiological functions but may provide treatments for cancers in which STAT3 activity is dysregulated.

In this study, we demonstrate that Tip60 associates with STAT3. Tip60 acts as a co-repressor for STAT3 to regulate gene expression in cytokine signaling and exerts a repressive effect on gene expression in part through the recruitment of histone deacetylase 7. Thus, this study provides a novel mechanism involved in the regulation of STAT3 activity.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Reagents and Antibodies-- Anti-Myc (9E10) and anti-phospho-tyrosine (PY99) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-FLAG (M2) and anti-phopho-STAT3 (Y704) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-STAT3 antibody was from R&D Systems, Inc. (Minneapolis, MN). Anti-HA antibody was from BAbCo (Richmond, CA). Tricostatin A (TSA) was from Sigma. Murine IL-9 and IL-6 were from R&D Systems Inc.

Plasmid Construction-- Tip60 N- and C-terminal deletion mutants were generated by PCR and confirmed by sequencing. The 5' primer used for C-terminal deletion mutants is 5'-ATGGACATCAGTGGCCGG-3'. For N-terminal deletion mutant N1, the 5' primer is 5'-TCCCCGTACCCACAG-3'. The 3' primers used for Tip60 deletion mutants are 5'-CACGGCTTGAGGCG-3' (for Tip60C3); 5'-ATTGTAGTCTTCCGTTG-3' (for Tip60C2); 5'-TTGATGCTGGTGAT-3' (for Tip60C1); and 5'-AGTGTCTGGTCACC-3' (for Tip60N1). Tip60 deletion mutants were cloned into pFLAG-CMV2 vector at EcoRI and BamHI sites. Tip60HAT- was obtained by PCR using pcDNA3.1 plasmid containing Tip60HAT- (15) as the template and cloned into pMT2 vector with a Myc tag at the EcoRI site.

Cell Culture and Transfection-- Human embryonic kidney (HEK) 293 cells and hepatoma HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Transient transfection was carried out by calcium phosphate co-precipitation. T lymphoma TS1 cells were maintained in Click's medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS and murine IL-9 (0.1 ng/ml). Electroporation was performed to establish stable cell lines expressing wild-type or mutant forms of Tip60 as described previously (43).

Cytokine Stimulation, Immunoprecipitation, and Immunoblotting-- TS1 cells were serum-starved in Dulbecco's modified Eagle's medium in the absence of IL-9 for 2-6 h. Cells (2×107/ml) were then stimulated with IL-9 (50 ng/ml) at 37 °C and lysed in 1 ml of TNE lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% glycerol, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium vanadate) containing 1% Nonidet P-40 for immunoprecipitation or 0.5% Nonidet P-40 for co-immunoprecipitation. Solubilized proteins were collected for immunoprecipitation and immunoblotting according to procedures described previously (44). All of the experiments were repeated at least twice with similar results.

Chloramphenicol Acetyltransferase (CAT) Assay-- HepG2 cells were transfected with 0.5 µg of pCMV-HA-STAT3, 2 µg of p4×SIE CAT (45) (or pCAT as a negative control), 0.2 µg of pCMV-beta -galactosidase in combination with different amounts of plasmids encoding wild-type or mutant Tip60, or pCMX-HA-HDAC7. Following 24 h of transfection, medium containing DNA-calcium phosphate precipitates was replaced by fresh medium and cells continued to grow for 24 h. Cells were washed twice with phosphate-buffered saline and then incubated in FBS-deprived medium with or without IL-6 (10 ng/ml). After 16 h, cells were harvested and the CAT assay was performed at 37 °C for 1 h in a 125-µl reaction containing 5 µl of n-butyryl-CoA (5 mg/ml) and 3 µl of D-threo-[dichloroacetyl-1-14C]chloramphenicol (CFA754, 54 mCi/mmol, 25 µCi/ml, Amersham Biosciences). Reactions were stopped by the addition of 300 µl of TMPD-xylene (2:1) and vortexed vigorously for 30 s. After centrifugation for 5 min, 10 µl of upper phase solution from each sample was taken for scintillation counting. The beta -galactosidase assay was performed to normalize the CAT activity.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tip60 Possesses Transcriptional Repression Activity-- Tip60 possesses intrinsic HAT activity and enhances the activity of transcriptional activators, supporting its role as a co-activator (7, 13, 14). However, some studies argue that Tip60 may act as a co-repressor for certain transcriptional regulators (15, 16, 46). In addition, yeast homologues of Tip60, SAS2, and SAS3 are involved in gene silencing (47, 48). Here, transient transfection assays were performed to test whether Tip60 can repress basal transcription in HEK 293 cells. In these experiments, we used pMH100-TK-LUC, which contains a luciferase gene driven by a thymidine kinase promoter with four copies of Gal4 binding site as a reporter plasmid (49). When pM-Tip60, which expresses Tip60 in-frame with the Gal4 DNA-binding domain (DBD), was co-transfected into HEK 293 cells with pMH100-TK-LUC, Tip60 repressed reporter gene expression in a dose-dependent manner (Fig. 1A). Overexpression of Gal4DBD-Tip60HAT-, a Tip60 HAT-deficient mutant with substitutions of two amino acids in the acetyl-CoA motif (15), also modestly repressed transcriptional activity (Fig. 1A).


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Fig. 1.   Tip60 represses basal transcription. Repressive effect of wild-type or mutant Tip60 in HEK 293 (A), HepG2 (B), or DF1 (C) cells. Various combinations of plasmids were co-transfected into HEK 293, HepG2, or DF1 cells in 6-well plates by calcium-phosphate method. Transfection mixture was replaced with fresh medium after transfection for 12 h. Following transfection for 36-48 h, cell lysates were prepared and the luciferase assay was performed with dual-luciferase assay kit (Promega) according to the manufacturer's manual. RLU, relative luciferase units.

Tip60 acts as a co-activator for Tat in HeLa cells but as a co-repressor for Tat in Jurkat cells (16). To examine the repressive effect of Tip60 in other cell lines, we co-transfected pM, pMTip60, or pMTip60HAT- together with pMH100-TK-LUC into HepG2 or DF1 cells. As shown in Fig. 1B, wild-type and HAT-Tip60 repressed transcription in HepG2 cells. Tip60 repressed reporter gene expression at a greater extent (up to 8-fold) in DF1 cells (Fig. 1C, columns 2-5), suggesting that the repressive effect of Tip60 is cell type-specific. In DF1 cells, the repressive effect of HAT-Tip60 was less than that of wild-type Tip60 (Fig. 1C, columns 6-9).

Histone Deacetylase 7 Potentiates the Repressive Effect of Tip60-- One of the mechanisms by which repressors or co-repressors regulate gene expression is through the recruitment of histone deacetylases (18). To test whether Tip60 can recruit other repressive molecules such as HDACs to regulate gene expression, we included the class I histone deacetylase member, HDAC1, and class II histone deacetylase member, HDAC7, in transient transfection assays. Co-transfection of pCMX-HA-HDAC7 together with pMTip60 greatly reduced reporter gene expression (Fig. 2A, columns 9-12) compared with co-transfection of pCMX-HA-HDAC7 with pM (Fig. 2A, column 8). Thus, HDAC7 potentiates the repressive effect of Tip60 in a dose-dependent manner. Unlike HDAC7, HDAC1 did not increase the repressive effect of Tip60 (Fig. 2A, columns 3-7), indicating that HDAC1 and HDAC7 have distinct roles in the regulation of Tip60-mediated transcriptional repression.


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Fig. 2.   HDAC7 potentiates the repressive effect of Tip60. A, co-transfection of HDAC7 but not HDAC1 enhances the repressive effect of Tip60 in HEK 293 cells. pCMX-HA-HDAC7 or pCMX-HA-HDAC1 (0.1-1.2 µg) was co-transfected with pM-Tip60 (0.4 µg) or empty vector into HEK 293 cells in 6-well plates by calcium-phosphate method. Total DNA amount for each transfection was kept the same by adjusting the amount of empty vector. B, TSA abrogates HDAC7-mediated repressive effect on Tip60. Following transfection for 24 h, TSA was added to transfected cells and incubated overnight before harvest for luciferase assays. C, HDAC7 modestly potentiates the repressive effect of Tip60 in DF1 cells. RLU, relative luciferase units.

To test whether histone deacetylase activity is required for HDAC7 to potentiate the repressive effect of Tip60, we treated transfected cells with TSA, an inhibitor of class I and II histone deacetylases, for 12 h before harvesting cells for luciferase assays. As shown in Fig. 2B, TSA abrogated the ability of HDAC7 to enhance Tip60-mediated repression (Fig. 2B, columns 7-10). As in HEK 293 cells, overexpression of HDAC7 enhanced the repressive activity of Tip60 in DF1 cells (Fig. 2C).

Tip60 Interacts with HDAC7-- To test whether Tip60 and HDAC7 complex in cells, pCMX-HA-HDAC7 and pCMV2-FLAG-Tip60 were co-transfected or transfected together with an empty vector into HEK 293 cells. The lysates of transfected cells were immunoprecipitated with anti-HA or anti-FLAG antibodies. Immunoblots probed with anti-HA indicated that immunoprecipitation with anti-FLAG pulled down HA-tagged HDAC7 only when pCMV2-FLAG-Tip60 but not empty vector was present (Fig. 3A). By probing blots of anti-HA immunoprecipitates with anti-FLAG, it was also demonstrated that Tip60 and HDAC7 formed a complex (Fig. 3A).


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Fig. 3.   Tip60 interacts with HDAC7. A, co-immunoprecipitation of Tip60 and HDAC7 in HEK-293 cells. 5 µg of plasmids pFLAG-CMV2-Tip60 and pCMV-HA-HDAC7 was co-transfected or transfected alone into HEK 293 cells, and cell lysates were immunoprecipitated with anti-FLAG (2 µl) or anti-HA (5 µl). Immunoblotting was performed with anti-FLAG or anti-HA. B, schematic presentation of FLAG-tagged Tip60 deletion mutants. C, pCMV-HA-HDAC7 (5 µg) was co-transfected with FLAG-tagged wild-type or various deletion mutants of Tip60 in pFLAG-CMV2 (5 µg) or empty vector into HEK 293 cells by calcium-phosphate method. Transfected cells were incubated for 36-48 h before harvest, and cell lysates were subjected to immunoprecipitation with 5 µl of anti-HA. After extensive wash, immunoprecipitated proteins and whole cell lysates were separated by 10% SDS-PAGE. After proteins were transferred onto a PVDF membrane and immunoblotted with anti-FLAG, the membrane was stripped and re-blotted with anti-HA. D, interaction of Tip60 with HDAC7 mutants in mammalian two-hybrid assay. pM-Tip60 (0.1 µg) was co-transfected with various HDAC7 deletion mutants in pVP16 (0.1 µg) or pVP16 into HEK 293 cells in 24-well plates. A firefly luciferase gene driven by four tandem Gal4 binding sites and a TATA motif was used as a reporter. Following transfection for 36-48 h, cell lysates were prepared and the luciferase assay was conducted by dual luciferase assay kit. Firefly luciferase activity was normalized with Renilla luciferase activity. RLU, relative luciferase units.

To define the regions in Tip60 essential for association with HDAC7, Tip60 deletion mutants were generated and cloned into the pCMV2-FLAG vector (Fig. 3B). Plasmids encoding wild-type or mutant Tip60 were co-transfected into HEK 293 cells together with pCMX-HA-HDAC7. Anti-HA immunoprecipitates and whole cell lysates were resolved by SDS-PAGE, and immunoblots were probed first with anti-FLAG and then with anti-HA. Although the C-terminal deletion mutants C1 (amino acids 1-451) and C2 (amino acids 1-366) and the N-terminal deletion mutant N1 (amino acids 261-513) co-immunoprecipitated with HDAC7, the C-terminal deletion mutant C3 (amino acids 1-255) did not associate with HDAC7 (Fig. 3C). Because Tip60C3 is well expressed in whole cell lysates, we conclude that the region between amino acids 261 and 366 that contains a zinc finger motif is essential for Tip60 association with HDAC7. To map the interaction region for Tip60 in HDAC7, we performed mammalian two-hybrid assays. They showed that amino acids 241-533 in HDAC7 are essential for Tip60 association (Fig. 3D).

Tip60 Interacts with STAT3-- We previously showed that Tip60 binds to the IL-9R alpha -chain, suggesting that Tip60 may be involved in IL-9 signaling. STAT3 plays an essential role in IL-9-induced expression of primary response genes such as c-Myc and Cited2 (43, 50). To explore the possibility that Tip60 may regulate STAT3 activity, we examined the association between proteins by immunoprecipitation. pCMV2-FLAG-Tip60 was co-transfected into HEK 293 cells with an empty vector or a plasmid encoding Myc-tagged STAT3. Immunoprecipitation with anti-Myc followed by immunoblotting with anti-FLAG showed that Tip60 associates with STAT3 (Fig. 4A). Although Tip60N1-(261-513) interacts with STAT3, Tip60C1-(1-451) does not (Fig. 4A). This indicates that the C-terminal region of Tip60 (amino acids 452-513) is necessary for STAT3 association. To map the Tip60-interacting domain in STAT3, we co-transfected plasmids expressing FLAG-Tip60 and Myc-tagged wild-type or mutant STAT3 (Fig. 4B) into HEK 293 cells. Fig. 4, C and D, shows that the central region of STAT3, which contains the DNA binding domain, is necessary and sufficient for Tip60 association.


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Fig. 4.   Tip60 interacts with STAT3. A, Tip60 and STAT3 co-immunoprecipitate in HEK 293 cells. pcDNA-Myc-STAT3 (5 µg) was co-transfected with 5 µg of pFLAG-CMV2-Tip60, pFLAG-CMV2-Tip60C1, pFLAG-CMV2-Tip60N1, or empty vector into HEK 293 cells. Cell lysates were immunoprecipitated with 20 µl of anti-Myc and immunoblotted with anti-FLAG or anti-Myc. B, schematic illustration of Myc-tagged STAT3 mutants. C and D, interaction of Tip60 with STAT3 mutants in HEK 293 cells. pCMV-FLAG-Tip60 (5 µg) was co-transfected with various STAT3 mutants in pcDNA-Myc (5 µg) into HEK 293 cells. Cell lysates were immunoprecipitated with anti-Myc (20 µl) and immunoblotted with anti-FLAG or anti-Myc. E, 2 × 107 log-phase TS1/Myc-Tip60 cells were harvested and lysed before or after starvation for 3 h followed by IL-9 (50 ng/ml) stimulation for 30 min. Cell lysates were immunoprecipitated with anti-Myc (20 µl) or anti-STAT3 (5 µl). Immunoblots were probed with anti-STAT3 or anti-Myc. NT, normal treatment. F, TS1 cells (1 × 108) were deprived of serum and IL-9 for 3 h prior to stimulation by IL-9 (50 ng/ml) for 15 min. Cells were harvested, and cell lysates were immunoprecipitated with 5 µl of anti-Tip60. Immunoblots were probed with anti-phosphotyrosine, anti-STAT3, and anti-Tip60, sequentially.

To further characterize the Tip60-STAT3 interaction following cytokine stimulation, we generated an IL-9-dependent TS1 cell line that constitutively expresses Myc-tagged Tip60. Tip60-STAT3 complexes were in anti-Myc or anti-STAT3 immunoprecipitates (Fig. 4E, lane 3 or 4), further demonstrating that Tip60 complexes with STAT3 in cells. To test whether the Tip60-STAT3 interaction in TS1 cells is induced by IL-9, we examined their co-immunoprecipitation in IL-9-stimulated or unstimulated cells following serum starvation. The Tip60-STAT3 interaction is constitutive and unaffected by IL-9 induction (Fig. 4E, lanes 1 and 2). Because serum starvation promotes degradation of Tip60 (data not shown), a much lower level of Tip60 was recovered from serum-starved cells (Fig. 4E, lanes 1, 2, 5, and 6) than unstarved cells (Fig. 4E, lanes 3 and 7). Endogenous STAT3·Tip60 complexes were also detected in anti-Tip60 immunoprecipitates from IL-9-stimulated or unstimulated TS1 cells, further supporting the finding that their association is not phosphorylation-dependent (Fig. 4F).

Tip60 Represses the Transcriptional Activity of STAT3-- To test whether Tip60 regulates STAT3 activity, a CAT reporter plasmid containing four tandem repeats of the STAT3 binding site (SIE) was transfected into HepG2 cells together with plasmids expressing beta -galactosidase, STAT3, and wild-type or mutant Tip60. Cell lysates from IL-6-stimulated or unstimulated cells were used for the CAT assay, which was normalized for beta -galactosidase activity. STAT3-mediated reporter gene expression was induced ~10-fold by IL-6. Overexpression of wild-type Tip60 attenuated the transcriptional activity of STAT3 in a dose-dependent manner, whereas the overexpression of Tip60C1, which does not interact with STAT3 (Fig. 4A), enhanced STAT3 activity (Fig. 5A). To further characterize the role of HDAC7 in Tip60-mediated repression of STAT3 transcriptional activity, we included pCMX-HDAC7 along with pCMV2-FLAG-Tip60 in these experiments and found that HDAC7 potentiates the repressive effect of Tip60 on STAT3-responsive reporter activity (Fig. 5B). In contrast, the overexpression of Tip60 could not repress the transcriptional activity of p53 (Fig. 5C) or Smad3 (data not shown), suggesting that Tip60 functions as a specific co-repressor for STAT3 through the recruitment of HDAC7.


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Fig. 5.   Tip60 acts as a co-repressor of STAT3 through the recruitment of HDAC7. A, Tip60 represses STAT3-driven reporter expression. B, co-transfection of HDAC7 potentiates the repressive effect of Tip60 on STAT3 activity. Various combinations of plasmids were co-transfected into HepG2 cells in 6-well plates, and the CAT assay was performed as described under "Materials and Methods." C, overexpression of Tip60 does not affect the transcriptional activity of p53. pG13-Luc (2 µg), a p53 specific reporter plasmid, was co-transfected into HepG2 cells with various combinations of pCMV2-p53, pCMV2-Tip60, and pCMX-HDAC7 as indicated in Fig. 5C by calcium-phosphate method. Following 24 h of transfection, medium containing DNA-calcium phosphate precipitates was replaced by fresh medium and cells continued to grow for 24 h. Cell lysates were prepared, and luciferase assay was performed with dual-luciferase kit. These experiments were repeated at least twice with similar results.

Tip60 Represses STAT3-mediated Primary Response Gene Expression following IL-9 Stimulation-- Because Tip60 regulates STAT3-driven reporter expression (Fig. 5), we tested whether Tip60 also regulates STAT3-mediated expression of primary response genes induced by IL-9. We established TS1 stable cell lines, which express Myc-tagged wild- type Tip60, Tip60HAT-, or Tip60C1 at comparable levels (Fig. 6A). Following serum starvation, total RNA were prepared from unstimulated or IL-9-stimulated cells. The samples were analyzed for the expression of c-myc, a target gene for STAT3 in cells transformed by Src or induced by IL-9 or platelet-derived growth factor (43, 51). As shown in Fig. 6B, c-myc was induced by IL-9 in parental TS1 cells, and ectopic expression of Tip60 or Tip60HAT- significantly attenuated c-myc induction following IL-9 treatment for 30 min. The difference of c-myc induction in Tip60 and Tip60HAT--transfected cells became significant 90 min following cytokine stimulation (Fig. 6C). Ectopic expression of Tip60C1 slightly increased c-myc expression in 30 and 90 min, which is consistent with the ability of Tip60C1 to up-regulate the STAT3-mediated reporter gene expression following IL-6 stimulation (Fig. 5A). These data suggest that Tip60C1 may function as a dominant-negative mutant for Tip60 to regulate STAT3 activity. Similar results were also obtained for Cited2 expression (data not shown), indicating that Tip60 down-regulates STAT3-induced primary response gene expression in IL-9-stimulated cells.


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Fig. 6.   Tip60 represses STAT3-mediated gene expression in IL-9-stimulated cells. A, schematic presentation and detection of Myc-tagged wild-type and mutant Tip60 expressed in TS1 cells. 2 × 107 log-phase TS1 cell lines stably transfected with wild-type or mutant Tip60 were harvested. Cell lysates were immunoprecipitated with anti-Myc, and immunoblot was probed with anti-Myc. B, overexpression of wild-type Tip60 represses IL-9-induced expression of c-myc. Cells were starved for 5 h prior to stimulation by IL-9 (50 ng/ml) for 30 or 90 min. Total RNA samples were isolated by TriZOL reagent from Invitrogen following manufacturer's instructions. 10 µg of total RNA of each sample was used for Northern blotting. The same nylon membrane was probed with c-myc and 36B4 sequentially. C, quantitation of c-myc expression. 36B4, a ribosome RNA-encoding housekeeping gene, was used as an internal control to normalize the expression of c-myc in TS1 cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we provide strong evidence that Tip60 acts as a co-repressor for basal and transcriptional activator-mediated gene expression, in part through the recruitment of HDAC7. The finding supports the notion that Tip60 and other MYST members may repress gene expression and provides a possible mechanism for the repressive effect of Tip60 on STAT3-mediated activation following cytokine stimulation. Recent studies have shown that Tip60 and HBO1, another MYST family member, regulate gene expression by acting as co-repressors (15, 16, 46). Because Tip60 possesses intrinsic HAT activity, it has been difficult to explain how Tip60 exerts its repressive function. Several studies have made it possible to begin to understand the co-repressor role of Tip60. First, although recombinant Tip60 acetylates histone subunits H2A, H3, and H4 in vitro using purified histones as substrates, the HAT activity of Tip60 is much weaker than that of p300 in the same assay (4). Second, a purified Tip60-containing nuclear multisubunit complex but not purified Tip60 alone promotes histone acetylation in nucleosomes (12), suggesting that Tip60 acetylates histones in chromatin when complexed with other accessory proteins such as TAP54alpha /beta , which possesses ATPase activity (12) and DNA helicase. However, TAP54alpha /beta and DNA helicase are not detectable in Tip60-containing complexes recruited to the KAI1 promoter via NFkappa B p50 (52), suggesting that Tip60 exerts its functions through interaction with different protein partners. Third, Tip60 may promote acetylation of cellular proteins other than histones to regulate gene expression. For example, Tip60 enhances the activity of wild-type but not an acetylation-deficient mutant of androgen receptor, demonstrating that Tip60 acts as a co-activator by promoting the acetylation of androgen receptor but not histones (53).

Our study shows that Tip60 interacts with HADC7 in cells, and co-transfection of HDAC7 potentiates the repressive effect of Tip60 in Gal4 DBD or STAT3 reporter assays. This raises the possibility that Tip60 may repress gene expression via complexes with HDAC7. Through association with transcriptional factors involved in diverse physiological functions in vivo, Tip60 may direct HDAC7 to modulate a subset of genes regulated by Tip60-associated transcription factors. Unlike class I HDACs, HDAC7 expression is tissue-specific (25, 54) and its localization is cell type-specific and signal-regulated (28, 54). These properties of HDAC7 may explain the cell type-specific effect of Tip60 on gene expression. Our study reveals that Tip60 possesses modest repressive activity in HEK 293 and HepG2 cells but potently represses transcription in DF1 cells. This suggests that the expression level and/or the localization of HDAC7 may be different in these cells and may explain the different effects of Tip60 on transcription in different cells (16, 46). In addition, the expression level and localization of Tip60 can be regulated by extracellular signals (9, 11, 15). In cell lines that poorly express HDAC7 or in which HDAC7 is cytoplasmic, Tip60 would tend to form complexes with other partners, which may result in positive regulation of gene expression. However, if the Tip60·HDAC7 complexes predominate over others, Tip60 may repress gene expression. Interestingly, the zinc finger region responsible for association with HDAC7 is highly conserved within MYST family members, indicating that similar mechanisms may mediate the repressive effect of other MYST family members.

In this study, we identified Tip60 as a STAT3-interacting protein. We show that Tip60 interacts with STAT3 in a phosphorylation-independent manner. Non-phosphorylated STAT3 is predominantly located in the cytoplasm. After phosphorylation by activated Jaks and other kinases, STAT3 forms dimers and translocates into nucleus (55). Tip60 is in the cytoplasm and nucleus in TS1 cells, and IL-9 stimulation induces the nuclear translocation of Tip60.2 This suggests that most Tip60·STAT3 complexes are in the cytoplasm prior to cytokine induction but are nuclear following stimulation. Tip60 exerts its repressive effect in a STAT3 reporter assay (Fig. 5A) and STAT3-mediated gene expression following IL-9 stimulation (Fig. 6B). Although overexpression of Tip60 only produces a 2-fold repression of the transcriptional activity of STAT3 in HepG2 cells, co-transfection of HDAC7 greatly enhances repression mediated by Tip60 (Fig. 5B). STAT3 and HDAC7 associate with the C terminus (Fig. 4A) and the central region (Fig. 3C) of Tip60, respectively, raising the possibility that Tip60 may recruit HDAC7 to STAT3 proteins. Therefore, Tip60 inhibits STAT3 activity through a mechanism distinct from that used by PIAS3, which inhibits STAT3 activity by reducing its DNA binding (42).

Consistent with the essential role of co-activators with HAT activity in transcription regulation, CBP/p300 and NCoA/SRC1a also play essential roles in STAT3-mediated gene expression (38, 39). Furthermore, the HAT activity of CBP/p300 is required for STAT3 activation (56). Interestingly, a STAT-interacting protein, Nmi, enhances STAT activity by increasing the association of p300 with STATs (57), supporting the essential role of p300 in regulating STAT activity. TSA treatment dramatically increases STAT3 activity induced by IL-6, indicating that HDACs are involved in regulation of STAT3 activity (56). Thus, HDAC7 recruited via Tip60 provides an efficient way to directly antagonize STAT3 activity conferred by the HAT activity of p300 or NCoA, probably through deacetylation of histones. However, it is also possible that Tip60-HDAC7 regulates the reversible acetylation of proteins other than histones. Tip60 and HDAC7 interact with endothelin-A in response to endothelin-1 induction (11), and it will be interesting to test whether endothelin-A is a substrate for these two proteins.

Although our study suggests that Tip60 represses gene expression mainly through the recruitment of HDAC7, we cannot exclude the possibility that other mechanisms are involved. Because the HAT activity of Tip60 is partially required for Tip60 to mediate basal and STAT3-mediated gene expression (Figs. 1C and 6C), Tip60 may acetylate histones and/or other cellular proteins to negatively regulate gene expression. SAS2, a yeast homologue of Tip60, acetylates H4-Lys-16 in gene silencing (58, 59). Chameau, a new Drosophila member of the MYST family, is required for the maintenance of Hox gene silencing and can partially substitute the SAS2 HAT in yeast (60). It is probable that the HAT of Tip60 plays a role in repressing gene expression through acetylation of histones. Because Tip60 promotes the acetylation of androgen receptor to regulate its transcriptional activity (53), it is also possible that Tip60 may acetylate transcription factors such as STAT3 to negatively regulate their transcriptional activity.

Our data suggest that Tip60 negatively regulates STAT3 activity, possibly in concert with PIAS3 and SOCS3. Dysregulation of STAT3 activity is involved in tumor development, and many studies are aimed at effective therapeutics to attenuate the signaling pathways involved. Whether dysregulated expression of Tip60 or HDAC7 is involved in abnormal activation of STAT3 in cancer has not yet been investigated. Our finding that a Tip60 C-terminal mutant loses the ability to repress STAT3 activity suggests that loss-of-function mutants of Tip60 may contribute to the development of disease states. Further studies will help in the development of Tip60 as a target for the treatment of various diseases including cancer.

    ACKNOWLEDGEMENTS

We thank Dr. Daniel Sliva for pMT2-Myc-Tip60; Dr. James Kamine for providing Tip60HAT- mutant construct; Dr. Heinz Baumann for pCAT and pSIECAT plasmids; Dr. Ronald M. Evans for pCMX-HA-HDAC7, pCMX-HA-HDAC1, and pMH100; and Dr. David Donner for reading the paper.

    FOOTNOTES

* This study was supported by National Institutes of Health Grants DK50570, CA78433, and HL48819 (to Y.-C. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the James T. Pardee-Carl A. Gerstacker Assistant Professor of Cancer Research Faculty Chair in Cancer Research at Case Western Reserve University Cancer Center.

|| To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Case Western Reserve University, 2109 Adelbert Rd., W353, Cleveland, OH 44106-4965. Tel.: 216-368-6931; Fax: 216-368-3395; E-mail: yxy36@po.cwru.edu.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210816200

2 H. Xiao, J. Chung, H.-Y. Kao, and Y.-C. Yang, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Tip60, Tat-interactive protein, 60 kDa; Esa, Essential sas2-related acetyltransferase; HAT, histone acetyltransferase; HIV, human immunodeficiency virus; CREB, cAMP-response element-binding protein; IL, interleukin; HDAC, histone deacetylase; SAS, Something About Silencing; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; STAT, signal transducers and activators of transcription; CBP, CREB-binding protein; HA, hemagglutinin; TSA, tricostatin A; HEK, human embryonic kidney; FBS, fetal bovine serum; SIE, STAT3 binding site; CAT, chloramphenicol acetyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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