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DEC1 Negatively Regulates the Expression of DEC2 through Binding to the E-box in the Proximal Promoter

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m300596200

1083-351X

Yuxin Li, Mingxing Xie, Xiulong Song, Sarah Gragen, Karuna Sachdeva, Yinsheng Wan, Bingfang Yan,

DNA Repair Mechanisms

Human DEC (differentiallyexpressed in chondrocytes), mouse STRA (stimulated with retinoic acid), and rat SHARP (split and hairyrelated protein) proteins constitute a new and structurally distinct class of the basic helix-loop-helix proteins. In each species, two members are identified with a sequence identity of >907 in the basic helix-loop-helix region and ∼407 in the total proteins, respectively. Recently, we have reported that DEC1 is abundantly expressed in colon carcinomas but not in the adjacent normal tissues. The present study was undertaken to extend the expression study of DEC1 and to determine whether DEC1 and DEC2 had similar expression patterns among paired cancer-normal tissues from the colon, lung, and kidney. Without exceptions, DEC1 was markedly higher in the carcinomas, whereas the opposite was true with DEC2. In stable transfectants, tetracycline-induced expression of DEC1 caused proportional decreases in the expression of DEC2. Co-transfection with DEC1 repressed the activity of a DEC2 promoter reporter by as much as 907. The repression was observed with wild type DEC1 but not its DNA binding-defective mutants. Studies with deletion and site-directed mutants located, in the proximal promoter, an E-box motif that supported the DEC1-mediated repression. Disruption of this E-box markedly abolished the ability of the reporter to respond to DEC1. Our findings assign for DEC1 the first target gene that is regulated through direct DNA binding. DEC/STRA/SHARP proteins are highly identical in the DNA binding domain but much more diverse in other areas. DEC1-mediated repression on the expression of DEC2 provides an important mechanism that these transcription factors regulate the cellular function not only by modulating the expression of their target genes but also the expression of members within the same class. Human DEC (differentiallyexpressed in chondrocytes), mouse STRA (stimulated with retinoic acid), and rat SHARP (split and hairyrelated protein) proteins constitute a new and structurally distinct class of the basic helix-loop-helix proteins. In each species, two members are identified with a sequence identity of >907 in the basic helix-loop-helix region and ∼407 in the total proteins, respectively. Recently, we have reported that DEC1 is abundantly expressed in colon carcinomas but not in the adjacent normal tissues. The present study was undertaken to extend the expression study of DEC1 and to determine whether DEC1 and DEC2 had similar expression patterns among paired cancer-normal tissues from the colon, lung, and kidney. Without exceptions, DEC1 was markedly higher in the carcinomas, whereas the opposite was true with DEC2. In stable transfectants, tetracycline-induced expression of DEC1 caused proportional decreases in the expression of DEC2. Co-transfection with DEC1 repressed the activity of a DEC2 promoter reporter by as much as 907. The repression was observed with wild type DEC1 but not its DNA binding-defective mutants. Studies with deletion and site-directed mutants located, in the proximal promoter, an E-box motif that supported the DEC1-mediated repression. Disruption of this E-box markedly abolished the ability of the reporter to respond to DEC1. Our findings assign for DEC1 the first target gene that is regulated through direct DNA binding. DEC/STRA/SHARP proteins are highly identical in the DNA binding domain but much more diverse in other areas. DEC1-mediated repression on the expression of DEC2 provides an important mechanism that these transcription factors regulate the cellular function not only by modulating the expression of their target genes but also the expression of members within the same class. basic helix-loop-helix helix-loop-helix electrophoretic mobility shift assay reverse transcription The basic helix-loop-helix (bHLH)1 proteins are intimately associated with developmental events such as cell differentiation and lineage commitment (1Littlewood T.D. Evan G.I. Helix-Loop-Helix Transcription Factors. Oxford University Press, Oxford, UK1998: 1-48Google Scholar, 2Bissonnette R.P. McGahon A. Mahboubi A. Green D.R. J. Exp. Med. 1994; 180: 2413-2418Crossref PubMed Scopus (50) Google Scholar, 3Cronmiller C. Schedl P. Cline T.W. Genes Dev. 1988; 2: 1666-1676Crossref PubMed Scopus (113) Google Scholar, 4Nikoloff D.M. McGraw P. Henry S.A. Nucleic Acids Res. 1992; 20: 3253-3257Crossref PubMed Scopus (78) Google Scholar, 5Hirose K. Morita M. Ema M. Mimura J. Hamada H. Fujii H. Saijo Y. Gotoh O. Sogawa K. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 1706-1713Crossref PubMed Scopus (223) Google Scholar, 6Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1397) Google Scholar). The HLH domain in the bHLH motif is responsible for dimerization, whereas the basic region mediates DNA binding (1Littlewood T.D. Evan G.I. Helix-Loop-Helix Transcription Factors. Oxford University Press, Oxford, UK1998: 1-48Google Scholar). Based on sequence alignment and domain analysis, human DEC (differentially expressed in chondrocytes), mouse STRA (stimulated withretinoic acid), and rat SHARP (split and hairy relatedprotein) constitute a new and structurally distinct class of bHLH proteins (7Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollè P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, 8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar, 9Rossner M.J. Dörr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar, 10Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Arch. Biochem. Biophys. 1997; 336: 294-298Google Scholar). These proteins are distantly related toDrosophila Hairy and E(spl) as well as the mammalian homologues (e.g. HES) with the highest sequence identity (∼407) in the bHLH region (1Littlewood T.D. Evan G.I. Helix-Loop-Helix Transcription Factors. Oxford University Press, Oxford, UK1998: 1-48Google Scholar, 11Dawson S.R. Turner D.L. Weintraub H. Parkhurst S.M Mol. Cell. Biol. 1995; 15: 6923-6931Crossref PubMed Scopus (183) Google Scholar, 12Kokubo H. Lun Y. Johnson R.L. Biochem. Biophys. Res. Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar). Like Hairy/E(spl)/Hes, DEC/STRA/SHARPs contain an orange domain and a proline residue in the DNA binding domain. However, the proline is located 2 residues more toward the NH2 terminus (1Littlewood T.D. Evan G.I. Helix-Loop-Helix Transcription Factors. Oxford University Press, Oxford, UK1998: 1-48Google Scholar, 8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar). Another major structural difference on the functional domains is that DEC/STRA/SHARPs, unlike Hairy/E(Spl)/Hes proteins, lack the COOH-terminal WRPW tetrapeptide motif (13Fisher A.L. Ohsako S. Caudy M. Mol. Cell. Biol. 1996; 16: 2670-2676Crossref PubMed Scopus (311) Google Scholar). Through this sequence, Hairy/E(spl)/Hes recruit corepressor Groucho to the transcription regulatory complex (13Fisher A.L. Ohsako S. Caudy M. Mol. Cell. Biol. 1996; 16: 2670-2676Crossref PubMed Scopus (311) Google Scholar). Recruitment of Groucho is responsible for a vast array of biological activities of Hairy/E(spl)/Hes proteins including cellular differentiation and lineage commitment (14Giebel B. Campos-Ortega J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6250-6254Crossref PubMed Scopus (67) Google Scholar, 15Poortinga G. Watanabe M. Parkhurst S.M. EMBO J. 1998; 17: 2067-2078Crossref PubMed Scopus (209) Google Scholar, 16Chen G. Fernandez J. Mische S. Courey A. Genes Dev. 1999; 13: 2218-2230Crossref PubMed Scopus (355) Google Scholar, 17Hojo M. Ohtsuka T. Hashimoto N. Gradwohl G. Guillemot F. Hageyama R. Development. 2000; 127: 2515-2522PubMed Google Scholar, 18Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Two members of DEC/STRA/SHARP proteins are identified in each mammalian species studied with a sequence identity of >907 in the bHLH region and ∼407 in the total proteins, respectively (8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar). They exhibit an overlapping tissue distribution, and their expression is highly elevated in response to environmental stimuli (7Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollè P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, 8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Biochem. Biophys. Res. Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar, 9Rossner M.J. Dörr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar, 10Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Arch. Biochem. Biophys. 1997; 336: 294-298Google Scholar). In rats that undergo seizure induction by kainic acid, the levels of mRNA encoding SHARP1 or -2 are sharply increased within 1 h in the brain (9Rossner M.J. Dörr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar). In cultured human cells, both DEC1 and DEC2 are markedly induced in response to hypoxia (19Miyazaki K. Kawamoto T. Tanimoto K. Nishiyama M. Honda H. Kato Y. J. Biol. Chem. 2002; 277: 47014-47021Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Co-transfection experiments with promoter reporters have identified functional hypoxia response elements in both DEC1 and DEC2 genes. These elements show high affinity toward hypoxia-inducible factor-1α and -औ, providing a molecular explanation on the co-regulatory phenomena of DEC1 and DEC2 during hypoxia response (19Miyazaki K. Kawamoto T. Tanimoto K. Nishiyama M. Honda H. Kato Y. J. Biol. Chem. 2002; 277: 47014-47021Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Rapid induction of these proteins in response to environmental stimuli suggests that DEC/STRA/SHARPs are protective against detrimental conditions. In addition to a potential protective role against environmental stimuli, DEC/STRA/SHARPs have been implicated in cell differentiation (7Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollè P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar, 10Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Arch. Biochem. Biophys. 1997; 336: 294-298Google Scholar, 20Shen M. Yoshida E. Yan W. Kawamoto T. Suardita K. Koyano Y. Fujimoto K. Noshiro M. Kato Y. J. Biol. Chem. 2002; 277: 50112-50120Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), maturation of lymphocytes (21Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar), and regulation of molecular clock (22Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Nature. 2002; 419: 841-844Crossref PubMed Scopus (524) Google Scholar). In a cell culture system, mouse STRA13 promotes neuronal but represses mesodermal and endodermal differentiation (7Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dollè P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (218) Google Scholar). Consistent with the inductive effect on neuronal differentiation, rat SHARP proteins are abundantly expressed in a subset of mature neurons (9Rossner M.J. Dörr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell. Neurosci. 1997; 9: 460-475Crossref PubMed Scopus (109) Google Scholar). DEC1 has recently been shown to promote chondrocyte differentiation at the early and terminal stages (20Shen M. Yoshida E. Yan W. Kawamoto T. Suardita K. Koyano Y. Fujimoto K. Noshiro M. Kato Y. J. Biol. Chem. 2002; 277: 50112-50120Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). STRA13-deficient mice, although surviving to adulthood, develop autoimmune diseases accompanied by accumulation of spontaneously activated T and B cells (21Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 2: 1040-1047Crossref PubMed Scopus (151) Google Scholar). In addition, the mouse proteins are recently found to regulate the expression of biological clock regulator Per(22Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Nature. 2002; 419: 841-844Crossref PubMed Scopus (524) Google Scholar). Recently, we and other investigators have recently demonstrated that deregulated cell survival by DEC1 may have oncogenic significance. In paired samples, DEC1 is abundantly expressed in colon carcinomas but not in the adjacent normal tissues (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar). High levels of DEC1 transcript are also detected in an array of cancer cell lines derived from a wide range of organs (24Ivanova A.V. Ivanov S.V. Danilkovitch-Miagkova A. Lerman M.I. J. Biol. Chem. 2001; 276: 15306-15315Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Cells that lack the functional tumor suppressor VHL (von Hippel-Lindau) express higher levels of DEC1 (24Ivanova A.V. Ivanov S.V. Danilkovitch-Miagkova A. Lerman M.I. J. Biol. Chem. 2001; 276: 15306-15315Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Forced expression of DEC1 antagonizes serum deprivation-induced apoptosis and selectively inhibits the activation of procaspases (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar). These findings suggest that overexpression of DEC1 provides cells with an unusual survival mechanism and thus is oncogenic. The present study was undertaken to extend the expression study on DEC1 and to determine whether DEC1 and DEC2 displayed similar expression patterns among paired tumor-normal tissues from the colon, lung, and kidney. Without exceptions, DEC1 was expressed markedly higher in the carcinomas, whereas DEC2 was expressed markedly higher in the adjacent normal tissues. Forced expression of DEC1 sharply decreased the expression of DEC2 and markedly repressed the activity of a DEC2 promoter reporter. Co-transfection experiments with mutant reporters and electrophoretic mobility shift assay (EMSA) located, in the proximal promoter, an E-box that supports DEC1-mediated repression. These findings provide direct evidence that DEC1 negatively regulates the expression of DEC2, which is largely achieved through direct DNA binding to the E-box in the proximal promoter of DEC2. Tri-reagent, FLAG-cytomegalovirus vector, and anti-FLAG antibody were purchased from Sigma. The goat anti-rabbit-IgG conjugated with alkaline phosphatase or horseradish peroxidase and ECL substrate were from Pierce. Dulbecco's modified Eagle's medium, LipofectAMINE, and the ThermoScript I reverse transcription-coupled PCR kit were from Invitrogen. The Dual-Luciferase reporter assay system and DNA binding buffer were from Promega. Unless otherwise indicated, all other reagents were purchased from Fisher. Samples were collected from patients who underwent surgical resection for histologically confirmed adenocarcinoma. As paired controls, specimens from the adjacent, grossly normal tissues were harvested. The samples (12 pairs) were collected from the colon, kidney, and lung with four pairs from each organ. The age of the patients was between 23 and 68 with seven male and five female. The size of tumors was generally 2–5 cm in diameter, and the degree of differentiation of tumors was moderate or poor as determined by pathological examination. Samples were freshly processed for RNA isolation and protein extraction. Total RNA was isolated with a Tri-reagent as described previously (25Zhang H. LeCluyse E. Liu L. Hu M. Matoney L. Yan B. Arch. Biochem. Biophys. 1999; 368: 14-22Crossref PubMed Scopus (180) Google Scholar). For the preparation of protein extracts, tissues were homogenized in lysis buffer (20 mm Tris-HCl, pH 7.4, 17 Triton X-100, 17 sodium deoxycholate, 0.17 SDS, 0.2 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol). The homogenates were centrifuged at 12,000 × g for 30 min to remove any insoluble precipitates. The protocol of using human pathological tissues was reviewed by the Institutional Review Board. The expression of DEC1 and DEC2 in human tissues and cultured cells was primarily determined by RT-PCR experiments with a ThermoScript I kit. Total RNA (2 ॖg) was subjected to the synthesis of the first strand cDNA with an oligo(dT) primer and a ThermoScript reverse transcriptase. The reactions were incubated initially at 50 °C for 30 min and then at 60 °C for 60 min after additional reverse transcriptase was added. The cDNAs were then subjected to PCR amplification with cycling parameters as follows: 95 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 or 40 s for a total of 32 cycles. The primers for DEC1 amplification were 5′-GTCTGTGAGTCACTCTTCAG-3′ and 5′-GAGTCTAGTTCTGTTTGAAGG-3′. The primers for DEC2 amplification were 5′-CGCCCATTCAGTCCGACTTGGAT-3′ and 5′-TGGTTGATCAGCTGGACACAC-3′. The primers for औ-actin amplification were 5′-GTACCCTGGCATTGCCGACAGGATG-3′ and 5′-CGCAACTAAGTCATAGTCCGCCTA-3′. The PCR-amplified products were analyzed by agarose gel electrophoresis. A cDNA encoding the full-length DEC1 was isolated by a cDNA-trapping method (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar, 26Hu M. Yan B. Anal. Biochem. 1999; 266: 233-235Crossref PubMed Scopus (9) Google Scholar). Several DEC1 mutant constructs were prepared by PCR with the full-length DEC1 as the template. These mutants had a specific sequence deleted or one or more amino acids substituted. Some of the mutant constructs were prepared with the SPORT vector (the NH2-terminal truncated mutants), whereas others (the COOH-terminal truncated mutants) were prepared with the FLAG vector to facilitate immunodetection. In some cases, a Kozak sequence was introduced for effective translation initiation. The DEC2 promoter reporter was prepared with the pGL3-basic luciferase vector (Promega). Human genomic DNA was isolated from the placenta with a DNA extraction kit (Qiagen) according to the manufacturer's instruction. A genomic fragment (−1,888 to +11) was generated by PCR with 5′-AACAGATGAACTGAACGGACCG-3′ and 5′-CCTCAGTGCAGTGTTGAAAGTG-3′. This PCR fragment was ligated to the pGL3 vector. Deletion mutants of this reporter were prepared by endonuclease digestion followed by ligation or PCR. The DEC2 promoter reporter had two E-box motifs that probably interact with DEC1, and the studies with deletion mutants suggested that the E-box in the proximal region supports DEC1-mediated repression. In order to definitively establish such a role, site-directed mutagenesis was performed to substitute two of the six nucleotides. The mutant construct was prepared with a QuikChange site-directed mutagenesis kit (Stratagene). Complementary oligonucleotides (5′-GATGGTACGTTCCGAACGGGAGCTGGGTGCTGG-3′) were synthesized to target this region. To perform the substitutions, the primers were annealed to a DEC2 promoter reporter and subjected to a thermocycler for a total of 15 cycles. The resultant PCR-amplified constructs were then digested with DpnI to remove the nonmutated parent construct. The mutated PCR-amplified constructs were used to transform XL1-Blue. The same approach was used to prepare three DEC1 mutants that had single or double residues substituted in the DNA binding domain (P56A, R58P, or both). The general sequence for the site-directed mutagenic oligonucleotides was 5′-GAGACCTACAAATTGGCGCACCCGCTCATCGAGAAAAAGAG-3′ with the nucleotides in boldface type substituted individually or simultaneously. All mutated constructs were subjected to sequencing analysis to confirm the desired mutation being made without secondary mutations. Cells (293T) were plated in 24-well plates in Dulbecco's modified Eagle's medium supplemented with 107 fetal calf serum at a density of 1.6 × 105cells/well. Transfection was conducted by lipofection with LipofectAMINE according to the manufacturer's instructions. Transfection mixtures contained DEC1 or a mutant construct (100 ng), reporter plasmid (100 ng), and the pRL-TK Renilla plasmid (1 ng). If a DEC1-stable line was used, DEC1 or its mutant construct was omitted from the transfection mixture. The transfected cells were cultured for an additional 24 h, washed once with phosphate-buffered saline, and resuspended in passive lysis buffer (Promega). The lysed cells were subjected to two cycles of freezing/thawing. The reporter enzyme activities were assayed with a Dual-Luciferase reporter assay system. This system contained two substrates, which were used to determine the activity of two luciferases sequentially. The firefly luciferase activity, which represented the reporter gene activity, was initiated by mixing an aliquot of lysates (20 ॖl) with Luciferase Assay Reagent II. Then the firefly luminescence was quenched, and the Renillaluminescence was simultaneously activated by adding Stop & Glo reagent to the sample wells. The firefly luminescence signal was normalized based on the Renilla luminescence signal. In cases where the reading on the luciferase activity was too high, the lysates were diluted, and luciferase activities were then determined to minimize the interference on the reading of the Renilla luciferase activity. Cells (293T) were transfected with DEC1 or a mutant, and nuclear extracts were prepared with a nuclear extraction kit (Active Motif). In some cases, DEC1-stable transfected cells were used but cultured in the presence or absence of tetracycline to modulate the expression of transfected DEC1. Nuclear proteins (10 ॖg) were incubated with radiolabeled double-stranded oligonucleotides (5′-CGTTCCGCACGTGAGCTGGG-3′) in a final volume of 10 ॖl containing 1× DNA binding buffer. For competition experiments, nuclear extracts were first incubated with a 10- or 50-fold molar excess of cold probe and then mixed with the radiolabeled probe. Oligonucleotides with a disrupted E-box were also used in the competition assays. For supershift assays, the anti-DEC1 or an anti-FLAG antibody was added either before or after the nuclear extracts were incubated with the radiolabeled probe. The protein-DNA complexes were resolved in 67 PAGE and visualized by autoradiography. Western analyses were conducted as described previously (27Zhu W. Song L. Matoney L. LeCluyse E. Yan B. Drug Metab. Dispos. 2000; 28: 186-191PubMed Google Scholar). The anti-DEC1 antibody against the COOH-terminal peptide was described elsewhere (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar). Protein concentration was determined with BCA assay (Pierce) with bovine serum albumin as the standard. Data are presented as mean ± S.D. of at least four separate experiments, except where results of blots are shown, in which case a representative experiment is depicted in the figures. We have reported that DEC1 is abundantly expressed in colon carcinoma but not in the adjacent normal tissues (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar). The initial focus of the present study was to extend the expression study on DEC1 and to determine whether DEC1 and DEC2 shared similar expression patterns among paired cancer-normal tissues from the colon, kidney, and lung. RT-PCR experiments with primers specific to DEC1 and DEC2 were performed. As shown in Fig.1, without exceptions, the levels of DEC1 mRNA were markedly higher in the carcinomas, whereas the levels of DEC2 mRNA were markedly higher in the adjacent normal tissues. Between paired samples, the levels of औ-actin mRNA were comparable. The carcinoma-related increase in DEC1 expression was also detected by Western blot (top of each depicted figure), suggesting that mRNA levels are indicative of the overall expression of these two genes. The inversed expression patterns between DEC1 and DEC2 suggest that DEC1 negatively regulates the expression of DEC2 orvice versa. In order to directly test this possibility, DEC1-stable transfected lines were used to study the expression relationship between DEC1 and DEC2. Two clonal stable lines were included: one expressing DEC1 (wild type) and the other expressing DEC1-M, which lacked the DNA binding domain. The stable lines were prepared with 293T cells and the pcDNA6/TR-pcDNA4 expression system; therefore, the expression of DEC1 and DEC1-M was inducibly regulated by tetracycline as described previously (23Li Y. Zhang H. Xie M. Hu M. Ge S. Yang D. Wan Y. Yan B. Biochem. J. 2002; 367: 413-422Crossref PubMed Google Scholar). As expected, the addition of tetracycline caused a concentration-dependent increase on the levels of DEC1 as determined by Western blots (Fig.2A, top). Consistent with the inducible increase in the levels of DEC1 protein, the levels of DEC1 mRNA were proportionally increased (data not shown). In contrast to the increased expression of DEC1, the levels of DEC2 mRNA were proportionally decreased (Fig. 2A). However, such inversed expression patterns were observed only in the cells expressing wild-type DEC1 (Fig. 2A) and not the cells expressing the DEC1 mutant, although the levels of DEC1-M were markedly induced by tetracycline (Fig. 2B). The inability of DEC1-M to down-regulate the expression of DEC2 suggests that DEC1-mediated repression is achieved through a DNA-binding mechanism. In order to directly test this possibility, reporter experiments and EMSA were conducted. A DEC2 promoter reporter (pLuc-1888) was constructed to contain the basal promoter and other potential regulatory sequences of the DEC2 gene (−1,888 to +11). This region was chosen because it contained two E-box motifs that commonly serve as target sequences for bHLH transcription factors (1Littlewood T.D. Evan G.I. Helix-Loop-Helix Transcription Factors. Oxford University Press, Oxford, UK1998: 1-48Google Scholar). A series of 5′ deletion mutants of this reporter was also prepared and designed to specify the location of DNA sequence that is targeted by DEC1 (Fig.3A, left). Co-transfection experiments were conducted to test these reporters for their ability to support DEC1-mediated activity. The stable transfected line (wild-type DEC1 only) was transfected again with a reporter construct and cultured in the presence or absence of tetracycline to modulate the expression of DEC1. The pRL-TK Renilla plasmid was also included in the transfection mixture to normalize transfection efficiency. As described in Fig. 3A (right), the addition of tetracycline decreased the activity of the pLuc-1888 reporter by as much as 907. Similar repression was observed with the reporters that had the sequence deleted up to nucleotide −535. In contrast, reporter pLuc-125, which had a further deletion from nucleotide −535 to −125, simultaneously lost the basal transcription activity and the ability to respond to DEC1, suggesting the importance of this region (−535 to −125) in both basal and regulatory transcription. We next examined whether responsiveness to DEC1 could be separated from the basal transcription activity in the DEC2 promoter reporter. Given the fact that this region (−535 to −125) contains a single E-box that is probably targeted by DEC1, a reporter with this E-box disrupted was tested for the ability to confer basal transcription. Reporter pLuc-535 was subjected to site-directed mutagenesis to selectively disrupt the E-box (CACGTG to AACGGG). Similarly, co-transfection experiments were performed. As shown in Fig.3A (bottom), disruption of this E-box (pLuc-535-M) caused little change in the basal activity (cultured without tetracycline), suggesting that this E-box contributes little to basal transcription. In contrast, the reporter mutant (pLuc-535-M) exhibited only ∼357 repression in response to DEC1 (Fig.3A, lane 8), which contrasts strikingly with 907 repression observed with the corresponding nonmutagenic reporter (Fig. 3A, lane 5). These findings suggest that the proximal E-box is largely responsible for DEC1-mediated repression. It should be emphasized that a similar observation was made with a substitution mutant reporter prepared from the longest reporter pLuc-1888, and the expression levels of DEC1 were comparable among all cells as determined by Western blots (data not shown). We next examined whether this E-box interacted directly with DEC1. The DEC1-stable line was cultured in the presence or absence of tetracycline, and nuclear extracts were prepared. Double-stranded oligonucleotides harboring this E-box were synthesized and radiolabeled. The labeled probe was incubated with the nuclear extracts and analyzed by EMSA. As shown in Fig. 3B, incubation with the extracts from the cells cultured in the presence of tetracycline yielded a shifted band (lane 8). This band was not detected when incubation was performed with the extracts from the cell cultured without tetracycline (lane 7). The shifted band was competed completely by 50× (lane 1) or partially by 10× excess cold probe (lane 3). However, the oligonucleotides (50×) that harbored a mutated E-box (E-box-M) showed no competitive activity (lane 2). In addition, the shifted band was supershifted by the anti-DEC1 but not