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E2A/HEB and Id3 Proteins Control the Sensitivity to Glucocorticoid-induced Apoptosis in Thymocytes by Regulating the SRG3 Expression

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m402145200

ISSN

1083-351X

Autores

Myunggon Ko, Jeongeun Ahn, Changjin Lee, Heekyoung Chung, Sung Ho Jeon, Hee Yong Chung, Rho Hyun Seong,

Tópico(s)

NF-κB Signaling Pathways

Resumo

The E protein family transcription factors encoded by the E2A and HEB genes are known to play critical roles in the coordinate regulation of lymphocyte development. Previous studies have shown that T cell receptor (TCR) signals rapidly induce Id3, a dominant negative antagonist of E2A activity and allow thymocytes to survive selection events in the thymus. Here we show that SRG3 acts as a novel downstream target of E2A/HeLa E box-binding (HEB) complex and modulates glucocorticoid (GC) susceptibility in thymocytes in response to TCR signals. We have identified a putative E box element in the SRG3 promoter that is required for optimal promoter activity. The transcription factors E2A and HEB specifically associate with the E box element. Moreover, E2A-HEB heterodimers cooperated to activate SRG3 transcription, which was inhibited by the expression of Id proteins. TCR-mediated signals rapidly induced Id3 via MEK/ERK activation and thereby kept the E2A/HEB complex from binding to the E box element in the SRG3 promoter. Retroviral transduction of Id3 also repressed the SRG3 expression by inhibiting the E box binding activity of the E2A/HEB complex. Intriguingly, enforced Id3 expression conferred thymocyte resistance to GCs, which could be overcome by the overexpression of SRG3. Taken together, these results suggest that Id3 may enhance the viability of immature thymocytes by at least rendering them resistant to GCs through SRG3 down-regulation. The E protein family transcription factors encoded by the E2A and HEB genes are known to play critical roles in the coordinate regulation of lymphocyte development. Previous studies have shown that T cell receptor (TCR) signals rapidly induce Id3, a dominant negative antagonist of E2A activity and allow thymocytes to survive selection events in the thymus. Here we show that SRG3 acts as a novel downstream target of E2A/HeLa E box-binding (HEB) complex and modulates glucocorticoid (GC) susceptibility in thymocytes in response to TCR signals. We have identified a putative E box element in the SRG3 promoter that is required for optimal promoter activity. The transcription factors E2A and HEB specifically associate with the E box element. Moreover, E2A-HEB heterodimers cooperated to activate SRG3 transcription, which was inhibited by the expression of Id proteins. TCR-mediated signals rapidly induced Id3 via MEK/ERK activation and thereby kept the E2A/HEB complex from binding to the E box element in the SRG3 promoter. Retroviral transduction of Id3 also repressed the SRG3 expression by inhibiting the E box binding activity of the E2A/HEB complex. Intriguingly, enforced Id3 expression conferred thymocyte resistance to GCs, which could be overcome by the overexpression of SRG3. Taken together, these results suggest that Id3 may enhance the viability of immature thymocytes by at least rendering them resistant to GCs through SRG3 down-regulation. Gene expression that occurs during thymocyte development is tightly controlled by coordinated actions of a variety of transcription factors (1Clevers H. Ferrier P. Curr. Opin. Immunol. 1998; 10: 166-171Crossref PubMed Scopus (31) Google Scholar, 2Osborne B.A. Curr. Opin. Immunol. 2000; 12: 301-306Crossref PubMed Scopus (22) Google Scholar). Among them, the E proteins have been suggested as one of the key regulators of proper B and T lymphogenesis (3Bain G. Robanus Maandag E.C. te Riele H.P. Feeney A.J. Sheehy A. Schlissel M. Shinton S.A. Hardy R.R. Murre C. Immunity. 1997; 6: 145-154Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 4Bain G. Murre C. Semin. Immunol. 1998; 10: 143-153Crossref PubMed Scopus (78) Google Scholar, 5Bain G. Maandag E.C. Izon D.J. Amsen D. Kruisbeek A.M. Weintraub B.C. 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The E proteins, also known as class I helix-loop-helix (HLH) 1The abbreviations used are: HLH, helix-loop-helix; bHLH, basic HLH; GR, glucocorticoid receptor; TCR, T cell receptor; GC, glucocorticoid; SP, single-positive; DP, double-positive; DN, double negative; PMA, phorbol 12-myristate 13-acetate; Dex, dexamethasone; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; FBS, fetal bovine serum; 2-ME, 2-mercaptoethanol; HEB, HeLa E box-binding; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; EGFP, enhanced green fluorescent protein. 1The abbreviations used are: HLH, helix-loop-helix; bHLH, basic HLH; GR, glucocorticoid receptor; TCR, T cell receptor; GC, glucocorticoid; SP, single-positive; DP, double-positive; DN, double negative; PMA, phorbol 12-myristate 13-acetate; Dex, dexamethasone; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; FBS, fetal bovine serum; 2-ME, 2-mercaptoethanol; HEB, HeLa E box-binding; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; EGFP, enhanced green fluorescent protein. proteins, were originally identified as factors that specifically bind with high affinity to the palindromic CANNTG sequence, referred to as the E box element in the Ig enhancer (12Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1363) Google Scholar, 13Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (335) Google Scholar, 14Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar). The mammalian E proteins consist of E2A, E2-2, and HeLa E box-binding (HEB) proteins (15Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (405) Google Scholar, 16Glimcher L.H. Singh H. Cell. 1999; 96: 13-23Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 17Hu J.S. Olson E.N. Kingston R.E. Mol. Cell. Biol. 1992; 12: 1031-1042Crossref PubMed Scopus (251) Google Scholar). Although HEB and E2-2 are encoded by distinct genes, E2A gene encodes two E proteins, E12 and E47, through alternative RNA splicing (13Henthorn P. Kiledjian M. Kadesch T. Science. 1990; 247: 467-470Crossref PubMed Scopus (335) Google Scholar, 14Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar, 17Hu J.S. Olson E.N. Kingston R.E. Mol. Cell. Biol. 1992; 12: 1031-1042Crossref PubMed Scopus (251) Google Scholar, 18Sun X.H. Baltimore D. Cell. 1991; 64: 459-470Abstract Full Text PDF PubMed Scopus (316) Google Scholar). E proteins share a highly conserved domain called helix-loop-helix (HLH), which allows for dimerization between HLH family members (19Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). The conserved basic domain immediately NH2-terminal to HLH domain facilitates the binding of HLH dimers to the canonical E box element. All of the E proteins have the potential to interact with DNA in a dimeric configuration either as an E protein homodimer/heterodimer or, more commonly, as a heterodimer containing an E protein family member together with one of several cell type-specific bHLH proteins. Upon DNA binding, E proteins regulate the transcription of target genes through NH2-terminal transactivation domains (20Quong M.W. Massari M.E. Zwart R. Murre C. Mol. Cell. Biol. 1993; 13: 792-800Crossref PubMed Scopus (130) Google Scholar, 21Aronheim A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (104) Google Scholar, 22Massari M.E. Jennings P.A. Murre C. Mol. Cell. Biol. 1996; 16: 121-129Crossref PubMed Scopus (86) Google Scholar).Tissue-specific differential combinatorial associations between bHLH dimers play pivotal roles in imparting specificity and diversity in functional modulation of distinct E box transcriptional regulatory activities depending on the cell context. In thymocytes, the E2A/HEB heterodimer is known as a major basic HLH (bHLH), whereas E2A homodimer is predominant in B-lineage cells (23Sawada S. Littman D.R. Mol. Cell. Biol. 1993; 13: 5620-5628Crossref PubMed Scopus (154) Google Scholar, 24Shen C.P. Kadesch T. Mol. Cell. Biol. 1995; 15: 4518-4524Crossref PubMed Scopus (160) Google Scholar). By analyzing various knockout mice deficient in E protein member(s), it is well characterized that E2A and HEB activity is required at multiple stages of thymocyte maturation. Mice deficient in E2A or E47 exhibited hypocellularity in the thymus and a partial block at the CD44+CD25– (double-negative 1, DN1) stage (8Bain G. Engel I. Robanus Maandag E.C. te Riele H.P. Voland J.R. Sharp L.L. Chun J. Huey B. Pinkel D. Murre C. Mol. Cell. Biol. 1997; 17: 4782-4791Crossref PubMed Google Scholar, 9Bain G. Quong M.W. Soloff R.S. Hedrick S.M. Murre C. J. Exp. Med. 1999; 190: 1605-1616Crossref PubMed Scopus (110) Google Scholar). Targeted inactivation of HEB also led to thymic hypocellularity and a strong developmental block at the immature single-positive stage (10Barndt R. Dai M.F. Zhuang Y. J. Immunol. 1999; 163: 3331-3343PubMed Google Scholar). Mice expressing HEBbm, a dominant negative HEB that inhibits both E2A and HEB activity, displayed a developmental arrest at the CD44–CD25+ (DN3) stage (11Barndt R.J. Dai M. Zhuang Y. Mol. Cell. Biol. 2000; 20: 6677-6685Crossref PubMed Scopus (144) Google Scholar). However, for the proper progression at some checkpoints, E2A activity is negatively regulated by signals emanating from antigen receptors. For example, E2A deficiency allowed the developmental progression and proliferation of DN thymocytes that have failed β-selection (7Engel I. Johns C. Bain G. Rivera R.R. Murre C. J. Exp. Med. 2001; 194: 733-745Crossref PubMed Scopus (154) Google Scholar). E2A or E47 deficiency also promoted the positive selection of CD4+CD8+ double-positive (DP) thymocytes expressing the relevant αβTCR (8Bain G. Engel I. Robanus Maandag E.C. te Riele H.P. Voland J.R. Sharp L.L. Chun J. Huey B. Pinkel D. Murre C. Mol. Cell. Biol. 1997; 17: 4782-4791Crossref PubMed Google Scholar, 9Bain G. Quong M.W. Soloff R.S. Hedrick S.M. Murre C. J. Exp. Med. 1999; 190: 1605-1616Crossref PubMed Scopus (110) Google Scholar). In addition, E2A protein played positive regulatory roles in apoptosis and growth arrest, suggesting that inactivation of E2A may allow survival of thymocytes that would have otherwise died during thymic selection processes (25Engel I. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 996-1001Crossref PubMed Scopus (88) Google Scholar, 26Park S.T. Nolan G.P. Sun X.H. J. Exp. Med. 1999; 189: 501-508Crossref PubMed Scopus (60) Google Scholar, 27Peverali F.A. Ramqvist T. Saffrich R. Pepperkok R. Barone M.V. Philipson L. EMBO J. 1994; 13: 4291-4301Crossref PubMed Scopus (185) Google Scholar, 28Engel I. Murre C. EMBO J. 2004; 23: 202-211Crossref PubMed Scopus (92) Google Scholar).E protein activity can be controlled by heterodimerization with Id proteins referred to as class V HLH proteins (29Quong M.W. Romanow W.J. Murre C. Annu. Rev. Immunol. 2002; 20: 301-322Crossref PubMed Scopus (162) Google Scholar). Id proteins act as functional antagonists of E protein activity in a dominant negative manner since they do not bind to DNA because of the lack of a basic DNA-binding domain while efficiently forming highly stable heterodimers with selected E proteins through the HLH domain (15Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (405) Google Scholar, 16Glimcher L.H. Singh H. Cell. 1999; 96: 13-23Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 30Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1791) Google Scholar). Acting as functional antagonists of bHLH transcription factors, Id proteins have been shown to play pivotal roles as positive regulators of cell growth and negative regulators of cell differentiation (27Peverali F.A. Ramqvist T. Saffrich R. Pepperkok R. Barone M.V. Philipson L. EMBO J. 1994; 13: 4291-4301Crossref PubMed Scopus (185) Google Scholar, 30Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1791) Google Scholar, 31Kreider B.L. Benezra R. Rovera G. Kadesch T. Science. 1992; 255: 1700-1702Crossref PubMed Scopus (222) Google Scholar, 32Yan W. Young A.Z. Soares V.C. Kelley R. Benezra R. Zhuang Y. Mol. Cell. Biol. 1997; 17: 7317-7327Crossref PubMed Scopus (205) Google Scholar, 33Hara E. Yamaguchi T. Nojima H. Ide T. Campisi J. Okayama H. Oda K. J. Biol. Chem. 1994; 269: 2139-2145Abstract Full Text PDF PubMed Google Scholar, 34Iavarone A. Garg P. Lasorella A. Hsu J. Israel M.A. Genes Dev. 1994; 8: 1270-1284Crossref PubMed Scopus (337) Google Scholar, 35Sun X.H. Cell. 1994; 79: 893-900Abstract Full Text PDF PubMed Scopus (246) Google Scholar, 36Shoji W. Yamamoto T. Obinata M. J. Biol. Chem. 1994; 269: 5078-5084Abstract Full Text PDF PubMed Google Scholar, 37Lister J. Forrester W.C. Baron M.H. J. Biol. Chem. 1995; 270: 17939-17946Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). There are four Id protein members: Id1, Id2, Id3, and Id4 (30Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1791) Google Scholar, 38Christy B.A. Sanders L.K. Lau L.F. Copeland N.G. Jenkins N.A. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1815-1819Crossref PubMed Scopus (303) Google Scholar, 39Deed R.W. Bianchi S.M. Atherton G.T. Johnston D. Santibanez-Koref M. Murphy J.J. Norton J.D. Oncogene. 1993; 8: 599-607PubMed Google Scholar, 40Riechmann V. van Cruchten I. Sablitzky F. Nucleic Acids Res. 1994; 22: 749-755Crossref PubMed Scopus (250) Google Scholar). Among them, Id2 and Id3 are known to be abundantly expressed in B and T lymphocytes (41Kee B.L. Quong M.W. Murre C. Immunol. Rev. 2000; 175: 138-149Crossref PubMed Google Scholar, 42Rivera R.R. Johns C.P. Quan J. Johnson R.S. Murre C. Immunity. 2000; 12: 17-26Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Genetic studies have demonstrated that Id3 is essential for proper thymocyte maturation in both class I- and class II-restricted TCR transgenic mice (42Rivera R.R. Johns C.P. Quan J. Johnson R.S. Murre C. Immunity. 2000; 12: 17-26Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Intriguingly, inactivation of both E47 and Id3 restored normal thymocyte maturation, which clearly indicates genetic interaction between them in the thymus (42Rivera R.R. Johns C.P. Quan J. Johnson R.S. Murre C. Immunity. 2000; 12: 17-26Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Upon pre-TCR-mediated signaling in DN thymocytes, Id3 proteins were rapidly induced and thereby down-regulated E protein DNA binding to promote β-selection (7Engel I. Johns C. Bain G. Rivera R.R. Murre C. J. Exp. Med. 2001; 194: 733-745Crossref PubMed Scopus (154) Google Scholar). In addition, it has been suggested that Id3 expression by TCR activation of the Ras/MEK/ERK pathway also promotes the maturation of immature DP cells by inhibiting the E box binding activity of E2A (9Bain G. Quong M.W. Soloff R.S. Hedrick S.M. Murre C. J. Exp. Med. 1999; 190: 1605-1616Crossref PubMed Scopus (110) Google Scholar, 43Bain G. Cravatt C.B. Loomans C. Alberola-Ila J. Hedrick S.M. Murre C. Nat. Immun. 2001; 2: 165-171Crossref Scopus (226) Google Scholar). Taken together, these results imply that only the DP thymocytes that repress E2A activity by Id3 upon TCR/CD3 engagement may survive thymic selection and are able to differentiate into SP thymocytes. However, the specific nuclear target that is regulated by this interaction between E2A and Id3 protein at this transition has yet to be identified.Here we showed that SRG3 acts as a novel nuclear target whose expression is regulated by the E2A-Id3 interaction. E2A/HEB specifically bound to the E box element in the SRG3 promoter and activated its activity. Upon TCR-triggered signals, Id3 was rapidly induced and prevented the E2A/HEB activator complex from binding to the E box element in the SRG3 promoter, resulting in SRG3 down-regulation. Additionally, ectopic overexpression of Id3 by the retroviral transduction system decreased SRG3 expression by inhibiting E2A/HEB binding. It also rendered primary thymocytes and immature thymoma cells resistant to GC-triggered apoptosis by down-regulating the expression of SRG3. Taken together, these observations suggest that Id proteins may enhance the viability of DP thymocytes by rendering GC resistance by down-regulating SRG3 expression.EXPERIMENTAL PROCEDURESMice, Cells, Reagents, and Antibodies—Transgenic mice overexpressing SRG3 in the FVB background were described previously (44Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). The murine DP thymoma, 16610D9, was provided by Dr. C. Murre (University of California, San Diego, La Jolla, CA) and cultured in Opti-MEM (Invitrogen) containing 10% FBS supplemented with 50 μm 2-mercaptoethanol. NIH3T3 was maintained in Dulbecco's modified Eagle's medium containing 10% bovine calf serum.We purchased PD98059 from Calbiochem; phorbol 12-myristate 13-acetate (PMA), A23187 (ionomycin), cycloheximide (CHX), and dexamethasone (Dex) from Sigma; anti-actin (sc-1615) and anti-GR (M-20) antibodies from Santa Cruz Biotechnology; and fluorescein isothiocyanate-conjugated (556419) or biotin-conjugated (556418) annexin V, quantum red-conjugated streptavidin (S-2899), and anti-Fas (554254) antibodies from Pharmingen. Antiserum against SRG3 was raised from rabbits in our laboratory as described previously (44Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar).Nuclear Extracts Preparation and Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared as described previously (45Kim D. Xu M. Nie L. Peng X.C. Jimi E. Voll R.E. Nguyen T. Ghosh S. Sun X.H. Immunity. 2002; 16: 9-21Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). EMSA was performed as follows. Double-stranded oligonucleotides were end-labeled with [γ-32P]ATP by using T4 polynucleotide kinase followed by purification over a MicroSpin™ G-25 column (Amersham Biosciences). The DNA binding reaction was performed at room temperature for 30 min in a total volume of 20 μl containing nuclear extracts (2–5 μg), an appropriate labeled probe, and 2 μg of poly(dI-dC) in binding buffer containing 20 mm HEPES (pH 7.9), 40 mm KCl, 2.5 mm MgCl2, 1 mm dithiothreitol, and 5% glycerol. The resulting protein-DNA complexes were resolved in a nondenaturing 5% polyacrylamide gel in 0.5× TBE buffer. Gels were visualized by autoradiography on dry gel. The sequences of each oligonucleotide used for EMSA are as follows: E84, 5′-AGGAGGTGGCATCTGCGCGCGCGG-3′; μE5, 5′-TCGAAGAACACCTGCAGCAGCT-3′; and Oct-1, 5′-TGTCGAATGCAAATCACTAGAA-3′.Northern Blot Analysis and Reverse Transcriptase (RT)-PCR—Total RNA was prepared from cells by resuspension in TRIzol® reagent (Invitrogen) according to the manufacturer's instruction. Total RNA (5–10 μg) was separated on a 1.2% formaldehyde gel and then blotted to Hybond™-N (Amersham Biosciences). Northern blots were probed with Id3 and SRG3 probes. The 405-bp Id3 probe was generated by random priming using the EcoRI fragment of Id3/S-003 vector, and the specific probe for SRG3 was generated as described previously (46Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). RT-PCR analysis was performed as described previously (45Kim D. Xu M. Nie L. Peng X.C. Jimi E. Voll R.E. Nguyen T. Ghosh S. Sun X.H. Immunity. 2002; 16: 9-21Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).Plasmids, Mutagenesis, Transfection, and Reporter Assay—The pSRG3-Luc reporter construct was described previously (46Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). pHBAneo-E47 and pHBAneo-E12 were from Dr. C. Murre (University of California, San Diego, La Jolla, CA). For construction of pCAGGS-E47, the BamHI fragment of pHBAneo-E47 was cloned into pBluescript and then the XbaI/EcoRI fragment was inserted into pCAGGS vector. pCAGGS-E12 was constructed by ligating the BamHI fragment of pHBAneo-E12 with BglII-digested pCAGGS vector. HEB cDNA was amplified by RT-PCR and cloned into pGEM®-T Easy vector, and then the XhoI fragment was inserted into pCAGGS vector. The PCR primers for construction of pCAGGS-HEB are as follows: 5′-GTGGCTCGAGATGAATCCCAGCA-3′ and 5′-CTGGCTCGAGTTTACAGATGACCCAT-3′. Mutagenesis of the E box sequences (CATCTG into CTGCAG) in the SRG3 promoter, transient transfection, and luciferase assay were performed as described previously (46Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). The PCR primers for the mutagenesis of E84 element are as follows: 5′-AGGAGGTGGCTGCAGCGCGCGCGG-3′ and 5′-CCGCGCGCGCTGCAGCCACCTCCT-3′.Retroviral Transduction—The retroviral vector LZRSpBMN-linker IRES-EGFP (NotI–) (S-003) and Id3/S-003 was from Dr. C. Murre (University of California, San Diego, La Jolla, CA). The ϕNX-eco packaging cell line was transfected with S-003 or Id3/S-003 vector by calcium phosphate precipitation and incubated in the presence of 2 μg/ml puromycin for 4 weeks. Retrovirus-containing supernatants were collected following incubation of the selected retroviral producer cells for 24 h and stored at –80 °C after filtering. For transduction, the retroviral supernatant (1 ml) was supplemented with Polybrene (8 μg/ml), incubated on ice for at least 10 min, and transferred to the standard 6-well tissue culture plates containing target cells (1 × 106 cells in 1 ml of fresh medium), followed by centrifugation at 2,800 rpm for 90 min at 32 °C. After centrifugation, growth medium was replaced with fresh medium. Cells were harvested and analyzed 36–48 h after infection. For the transduction of primary thymocytes, cells were pre-treated with 3 μg/ml concanavalin A (Vector Laboratories) for 2 days and spininfected as described.Cell Death Measurement—Single cell suspensions of thymocytes derived from 4- to 5-week-old wild-type or hCD2-SRG3+ Tg FVB mice were maintained in RPMI 1640 medium containing 10% FBS and 50 μm 2-mercaptoethanol supplemented with glutamine, penicillin, and streptomycin. A portion of apoptotic cells was assessed by annexin V staining. At the end of each treatment, cells were harvested and resuspended in biotin-conjugated annexin V, incubated at room temperature for 15 min, washed, resuspended in quantum red-conjugated streptavidin, and analyzed by flow cytometry (20,000–30,000 events) with the CellQuest™ software using FACStar (BD Biosciences).RESULTSOverexpression of Id3 Proteins Represses the SRG3 Gene Expression—In thymocytes, E2A protein mostly binds to the E box element with the consensus CANNTG sequence as a complex with the HeLa E box-binding (HEB) protein, another bHLH family transcription factor highly expressed in developing T cells (23Sawada S. Littman D.R. Mol. Cell. Biol. 1993; 13: 5620-5628Crossref PubMed Scopus (154) Google Scholar). We found that the putative E box element (designated E84) CATCTG exists at the –84 region in the SRG3 promoter and that it plays crucial roles in TCR signaling-mediated repression of the SRG3 gene expression to render thymocytes resistant to GC-mediated apoptosis (55Ko M. Jang J. Ahn J. Lee K. Chung H. Jeon S.H. Seong R.H. J. Biol. Chem. 2004; 279: 21903-21915Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In addition, the E2A/HEB protein complex is specifically associated with the putative E box element in the SRG3 promoter. It was suggested that expression of the E-protein inhibitor Id3 interferes with E-protein DNA binding activity because of the lack of the basic DNA-binding domain (29Quong M.W. Romanow W.J. Murre C. Annu. Rev. Immunol. 2002; 20: 301-322Crossref PubMed Scopus (162) Google Scholar). Therefore, it is conceivable that Id3 proteins negatively regulate SRG3 gene expression by antagonizing the binding of E2A/HEB to the E84 element in the SRG3 promoter. To examine the regulatory role of Id3 in modulating SRG3 expression, Id3 was retrovirally transduced into 16610D9 cells by using a retroviral vector, S-003. The Id3/S-003 retroviral vector allows translation of both FLAG-tagged mouse Id3 and enhanced green fluorescence protein (EGFP) from a bicistronic retroviral transcript (Fig. 1A). To transduce Id3 into 16610D9 cells, we used supernatants from ϕNX-eco retroviral packaging line into which Id3/S-003 or S-003 empty vector had been stably transfected. The transduction level was determined by flow cytometric analysis of transductants expressing EGFP (Fig. 1B). Subsequently, we assessed the level of SRG3 proteins in the whole-cell extracts from control and Id3-infected cells by immunoblotting. As shown in Fig. 1C, ectopic overexpression of Id3 reduced SRG3 protein expression by more than half, whereas the expression of GR and actin remained unchanged, suggesting that SRG3 gene expression is negatively controlled by Id3 expression. To determine whether the decrease in SRG3 protein levels upon Id3 transduction resulted from suppression of SRG3 transcription, we next proceeded to analyze the level of various transcripts in control and Id3-transduced cells by Northern blotting. As shown in Fig. 2A, mRNAs for FLAG-tagged Id3 whose expression is driven by the Id3 retroviral construct were detectable only in Id3-infected cells. As a result, the level of SRG3 mRNAs was reduced by approximately half (Fig. 2A), which was comparable with the decrease in SRG3 proteins upon Id3 transduction (Fig. 1C). The same result was reproduced by RT-PCR analysis showing repression of SRG3 transcription by Id3 overexpression, whereas the level of glyceraldehyde-3-phosphate dehydrogenase or hypoxanthine-guanine phosphoribosyltransferase control transcripts was not affected (Fig. 2B). Taken together, these results indicate that Id3 represses the SRG3 gene expression at the transcriptional level.Fig. 2Id3 transcriptionally represses SRG3 gene expression.A, Northern blot analysis. Equal amounts of total RNAs prepared from vector- and Id3-transduced 16610D9 cells were resolved on a 1.2% formaldehyde gel and then blotted to Hybond™-N (Amersham Biosciences). The Northern blots were probed with specific SRG3 and FLAG-Id3 probes. 18 S and 28 S rRNAs are shown. B, RT-PCR assay using cDNAs prepared from total RNAs of vector- or Id3-transduced 16610D9 cells. Each PCR amplification was carried out with primers specific to each transcript as labeled and in three different numbers of cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) served as controls.View Large Image Figure ViewerDownload (PPT)E2A/HEB Complex Activates the SRG3 Promoter Activity through the E84 Element, Which Is Inhibited by Id Proteins— Because Id3 was well known to repress the E box binding activity of E proteins in a dominant negative manner and E protein family members such as E47, E12, and HEB directly associated with the putative E box element in the SRG3 promoter (55Ko M. Jang J. Ahn J. Lee K. Chung H. Jeon S.H. Seong R.H. J. Biol. Chem. 2004; 279: 21903-21915Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), it is possible that E2A and HEB proteins act to activate SRG3 expression, and Id3 protein represses SRG3 transcription by preventing E2A/HEB binding to the E box element in the SRG3 promoter, leading to a decrease in SRG3 protein level. To clarify the contribution of the E2A/HEB complex to the activation of SRG3 gene expression, we generated pSRG3–210-Luc, a luciferase reporter construct driven by SRG3 proximal promoter encompassing the 210-bp nucleotides upstream of the SRG3 translation start site. Next, we measured the SRG3 reporter activity in NIH3T3 cells transiently transfected with combinations of E47, E12, or HEB expression vector. This cell line is appropriate for this assay because the SRG3 gene was expressed at low levels in the absence of E2A/HEB binding to the E box element (E84) in the SR

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