Functional Analysis of GC Box and its CpG Methylation in the Regulation of CYP1A2 Gene Expression

Drug Metab. Pharmacokinet. 24 (3): 269–276 (2009).

Note Functional Analysis of GC Box and its CpG Methylation in the Regulation of CYP1A2 Gene Expression Atsushi MIYAJIMA, Tomomi FURIHATA* and Kan CHIBA Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Although there is a putative GC box in the cytochrome P450 1A2 (CYP1A2) promoter, its function has remained undetermined. To understand the molecular mechanisms controlling CYP1A2 gene expression in the liver, we studied the roles of the GC box in promoter activity and the effects of its CpG methylation on CYP1A2 gene expression. The results of luciferase assays showed that promoter activity was significantly dependent on the presence of the intact GC box. The results of bisulfate sequencing showed that the CpG methylation status of the GC box was strongly associated with CYP1A2 mRNA expression in human cell lines and tissues, suggesting that CpG methylation is involved in the tissue-specific regulation of CYP1A2 gene expression. However, effects of in vitro CpG methylation of the GC box on the promoter activity were not so dramatic in the luciferase assay, suggesting that the major function of the methylated-CpG is not to inhibit transcription factors in binding to the GC box. Taken together, our results show that the GC box is a critical element for the CYP1A2 promoter and its epigenetic regulation mediated by CpG methylation may play important roles in the tissue-specific CYP1A2 gene expression. Genome-based approaches may be necessary for understanding this tissue-specific epigenetic mechanism. Keywords: CYP1A2 gene; GC box; CpG methylation; transcription factor network; epigenetic regulation

gene is predominantly expressed in the human liver whereas the CYP1A1 gene is ubiquitously expressed. Since the promoter region (－1 kbp from transcriptional start site) of the CYP1A2 gene only shares 42% nucleotide sequence homology with that of the CYP1A1 gene in contrast to the coding region, the functions of the CYP1A2 promoter likely contribute to the limited expression profile of the CYP1A2 gene in the liver. The high level of CYP1A2 gene expression in the liver is thought to be accomplished by two mechanisms controlling gene expression; one is a transcription factor network that makes it possible for the CYP1A2 gene to be efficiently transcribed in the liver, and the other is a chromatin structure that allows the network to appropriately work on the regulatory region of the CYP1A2 gene. As for the first mechanism, several studies have been carried out to identify the transcription factors regulating CYP1A2 gene expression in the liver. Hepatocyte nuclear factor 1a (HNF1a)4) and

Introduction Cytochrome P450 (CYP) 1A2 is a heme-containing monooxygenase that catalyzes the metabolism of various exogenous compounds such as flutamide and tizanidine.1) It is also known to be responsible for activation of carcinogenic chemicals.2) Therefore, CYP1A2 plays important roles in drug metabolism and in chemically induced carcinogenesis. CYP1A2 is also thought to have some physiological functions since this enzyme has been reported involved in the metabolism of endogenous compounds such as eicosanoids and steroids.3) The human CYP1A2 gene is located on chromosome 15 and shares the opposite-orientated 23 kbp of the 5?flanking region with the CYP1A1 gene. It belongs to the same gene family and, accordingly, the coding region of CYP1A2 mRNA shares 80% nucleotide sequence homology with that of CYP1A1 mRNA. However, the CYP1A2

Received; November 9, 2008, Accepted; November 30, 2008 *To whom correspondence should be addressed: Tomomi FURIHATA, Ph. D., Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan. Tel/Fax. ＋81-43-226-2894, E-mail: tomomif＠p.chiba-u.ac.jp This work was partially supported by a Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), MEXT, Japan.

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HNF4a,5) which are liver-enriched transcription factors, have been reported to enhance CYP1A2 gene expression by binding to the upstream enhancer region, which is likely to be important for the high level of CYP1A2 gene expression in the liver. In the promoter region, upstream stimulatory factor 1 (USF1) and nuclear factor-1 (NF-1) have been reported to bind to an E-box and an NF-1 like element, respectively, to regulate CYP1A2 promoter activity.6) In addition to these elements, a putative GC box proximal to the TATA box in the CYP1A2 promoter has also been identified, although its function in CYP1A2 gene transcription remains unknown.7) Recent studies show that the cell type-specific spatial arrangement of chromatin fiber and genomic DNA is a cornerstone for establishment of a tissue-specific gene expression profile.8) Epigenetic regulation, including DNA methylation and histone modifications, is responsible for this spatial arrangement to determine whether the transcription factor network(s) are allowed to interact with DNA to regulate gene expression. It is thought that DNA methylation plays a central role in epigenetic regulation, in which a 5?-position of cytosine in a 5?-CG-3? dinucleotide (CpG) is a target of DNA methylation in mammals.9) CpG methylation in the promoter region usually represses transcriptional activity of its gene by inhibiting transcription factors from binding to their cis-elements and/or by altering the chromatin structure.10) Since the CpG methylation pattern differs depending on the type of tissue,11) CpG methylation in the promoter region is thought to regulate tissue-specific gene expression as seen in the mouse lactate dehydrogenase c and human secretin receptor genes.12,13) The present study clarifies the roles of the GC box in the CYP1A2 gene expression in the liver. We first examined the function of the GC box in CYP1A2 promoter activity by luciferase assay in HepG2 cells. Then, we examined the association of DNA methylation status of CpG of the GC box with CYP1A2 mRNA expression in human cell lines and human tissues. The results suggest that the GC box is essential for CYP1A2 promoter activity and that a CpG site found in the GC box plays an important role in the epigenetic regulation of CYP1A2 gene in human tissue.

Materials and Methods Human cell lines and human tissues: HepG2 cells (hepatocellular carcinoma cells), Caco–2 cells (colorectal carcinoma cells) and HEK293 cells (human embryonic kidney cells) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-products, Woodland, CA), 50 U/ml of penicillin and 50 mg/ml of streptomycin (Invitrogen). HeLa cells (cervical adenocarcinoma cells) were cultured in RPMI1640 medium (Invitrogen) supplemented as de-

scribed above. All cells were cultured in the presence of 5% CO2 at 379C. Human tissues (liver, lung, small intestine and kidney) were supplied by the National Disease Research Interchange (Philadelphia, PA) through the Human and Animal Bridging Research Organization (Tokyo, Japan). Full permission for use of human tissues was obtained from the Ethics Committee of Graduate School of Pharmaceutical Sciences, Chiba University (Chiba, Japan) based on the Helsinki declaration. Cloning and plasmid construction: The region －3188/＋37 of the CYP1A2 gene (GenBank Accession No. AF253322) was amplified by PCR using specific primers, 5?-GGAACACAACGGGACTTCTTGGATGC-3? (forward) and 5?-GAGATTGGCAGGGTTGTAATGGCT -3? (reverse), and human genomic DNA extracted from human whole blood (Promega, Madison, WI) as a template. The PCR fragment was treated with T4 Polynucleotide Kinase (T4PNK, TaKaRa, Shiga, Japan) at 379C for 2 h according to the manufacturer's instructions. The fragment was inserted into the EcoRV site of the pGL4.17 vector (Promega) to generate a reporter construct named p4－3188/＋37. Four deletion constructs were generated by nested PCR using p4－3188/＋37 and the following forward primers: 5?-TATAAAAAGGCCACTCACCTAGAG-3? (p4 －28/＋37), 5?-GCTCTTCCTCATGTGTGCAGTGGG-3? (p4－342/＋37), 5?-CCCGAGTAGCTGGGATCACAGGC-3? (p4－1033/＋37) and 5?-ATGCTCTGTTTCTCTATTGGATTCCCC-3? (p4－1961/＋37). The reverse primer was the same as that used in amplification of the －3188/＋37 region. After T4PNK treatment, the 5?-deletion fragments were inserted into the pGL4.17 vector as described above. All deletion constructs were named as shown in parentheses. The wild-type GC box (5?-GGGCGG-3?) located at －37/－32 of the CYP1A2 promoter in p4－342/＋37 was mutated to 5?-GGGTAA-3? by site-directed mutagenesis, resulting in p4－342/＋37mt. Specific primers, 5?-ATCTGATAGGGGGTAATGTTTATAAAAAG-3? (forward) and 5?-CCCTATCAGATTGGCCTGGTTGTCCT3? (reverse), were designed for this mutagenesis. To avoid the effects of CpG methylation of the vector backbone on luciferase activity, we constructed reporter constructs using a CpG-free luciferase vector, pCpGLbasic vector, which was kindly provided by Dr. Rehli.14) The region －342/＋37 of the CYP1A2 promoter was amplified from p4－342/＋37 by PCR using the following primers: 5?-GCAagatctTGAGATTGGCAGGGTTGTAA-3? (forward) and 5?-CTctgcagGCTCTTCCTCATGTGTGCAGT-3? (reverse). The small letters indicate SpeI and HindIII recognition sites, respectively. After treatment with SpeI and HindIII, the fragment was inserted into the same sites of the pCpGL-basic vector to generate pC－342/＋37 as described above.

GC Box is Essential for Regulation of CYP1A2 Gene Expression

The human serum paraoxonase 1 (PON1, GenBank accession no. AF051133) gene was used as a positive control in the co-transfection assay.15) The region －1164/＋ 52 of the PON1 gene was amplified by PCR using specific primers, 5?-GGggtaccCTCTCCATATGTTCATGG-3? (forward) and 5?-TCCcccgggATAGACAAAGGGATCG3? (reverse). The small letters indicate KpnI and SmaI recognition sites, respectively. After treatment with KpnI and SmaI, the fragment was inserted into the same sites of pGL4.17 as described above, resulting in p4-PON1. An Sp1 mammalian expression vector (pCMVSp1) and Sp3 Drosophila expression vectors (pPacUsp3) were kindly provided by Dr. R. Tjian (Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA) and Dr. G. Suske (Philipps-Universitat, Marburg, Germany), respectively.16,17) An Sp1 Drosophila expression vector (pAcSp1) was generated by insertion of the Sp1 coding region of pCMVSp1 into a pAc5.1/V5-His vector (Invitrogen) as described previously.18) To generate an Sp3 mammalian expression vector, pPacUsp3 was treated with SphI and XhoI. The fragment was inserted into the pTARGETTM mammalian expression vector (pT, Promega) treated with same enzyme, resulting in Usp3/pT. All constructs were sequenced using a Dye Terminator Cycle Sequencing-Quick Start Kit (Beckman Coulter, Fullerton, CA) and a CEQ 2000 DNA Analysis System (Beckman Coulter) according to the manufacturer's instructions. Reverse transfections and luciferase assays in HepG2 cells: HepG2 cells were transfected with 200 ng/well of firefly luciferase reporter construct and 50 ng/well of pGL4.70 renilla luciferase expression vector (Promega) by reverse transfection using TransIT (Mirus, Madison, WT, USA). In brief, mixtures of DNA and TransIT were plated in a 48-well plate. Then HepG2 cells in complete growth medium were seeded in each well at a density of 3.6×104 cells/well. At 48 h after reverse transfection, firefly and renilla luciferase activities were measured using a Dual-LuciferaseTM Reporter Assay System (Promega) according to the manufacturer's instructions. Renilla luciferase activity of pGL4.70 was used to normalize firefly luciferase activity of the reporter construct. In co-transfection assays reporter constructs were co-transfected with 50 ng/well of an expression vector (pT, pCMVSp1 or USp3/pT) as described above. Luciferase assays in SL2 cells: Drosophila SL2 cells were grown at 249C in Drosophila Schneider's medium (Invitrogen) supplemented as described above. Transient transfection of SL2 cells was described in a previous report.18) In brief, SL2 cells were transfected with 100 ng/well of the firefly reporter construct, 50 ng/well of pGL4.70 renilla luciferase reporter vector and 50 ng/well of an expression vector (pAc5.1/V5-His, pAcSp1 or pPacUSp3) using Cellfectin reagent (Invitrogen). Lu-

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ciferase activity was determined using the same system as that described above. Gel mobility shift assay (GMSA): The methods used for GMSA were described in a previous report.18) In brief, probe double-stranded (ds) DNA was generated by annealing the complementary oligonucleotides containing the GC box of CYP1A2–48/–19 (5?-ATCTGATAGGGGGCGGTGTTTATAAAAAGG-3?). A GC box-mutated CYP1A2–48/–19 (5?-ATCTGATAGGGGGTAATGTTTATAAAAAGG-3?) and consensus oligonucleotides for the Sp1 dsDNA (5?-ATTCGATCGGGGCGGGGCGAGC-3?) were generated as described above. The sequences in parentheses are sense strands. CYP1A2–48/–19 was endlabeled with [g-32P]ATP (GE Healthcare, Piscataway, NJ) using T4PNK. Samples containing nuclear extracts from HepG2 cells were held for 20 min at room temperature with the probe and the mixtures were electrophoresed on polyacrylamide gels. Gels were dried and autoradiographed. Competition experiments were carried out as described above except that cold competitive dsDNA was added to the binding reaction mixture at a 50fold excess of the probe amount before addition of the probe. Total RNA extraction and reverse transcriptionpolymerase chain reaction (RT-PCR): Total RNA was isolated from human cell lines and human tissues by using a FastPureTM RNA kit (TaKaRa) according to the manufacturer's instructions. Isolated total RNA was then treated with RNase-free DNase I (TaKaRa) to remove contaminating genomic DNA. For RT-PCR, cDNA was generated by reverse transcription with random hexamers using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, NJ). After reverse transcription, CYP1A2 cDNA (forward, 5?GGACAGCACTTCCCTGAGAGTAGCG-3? and reverse, 5?-CAATCTTCTCCTGTGGGATGAGGTTG-3?) and glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH, forward, 5?-TGCACCACCAACTGCTTA-3? and reverse, 5?-GGATGCAGGGATGATGTTC-3?) were amplified by PCR with the primers shown in parentheses. Conditions of RT-PCR were as follows: 959C for 2 min and then 959 C for 15 s, 559C for 10 s and 729C for 30 s for 35 cycles and 729 C for 2 min for CYP1A2 and 959 C for 2 min and then 959C for 15 s, 509C for 10 s and 729C for 30 s for 25 cycles and 729C for 2 min for GAPDH. Genomic DNA extraction and bisulfite sequencing: Genomic DNA was extracted from human cell lines and human tissues using a FastPureTM DNA kit (TaKaRa) according to the manufacturer's instructions. It was treated with bisulfite using an EpitectTM Bisulfite kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The region －62/＋232 of the CYP1A2 gene containing four CpG sites was amplified from the genomic DNA by PCR using GoTaq Green

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Master Mix (Promega) and specific primers 5?-TAGGATAATTAGGTTAATTTGATAG-3? (forward) and 5?-AACTTAATCCAAACTACTCATTTAAAATC-3?(reverse). The primers were designed using MethPrimer online software (http://www.urogene.org/methprimer/).19) PCR conditions were as follows: 959C for 2 min and then 959C for 30 s, 479C for 45 s and 729 C for 45 s for 3 cycles, followed by 959C for 30 s, 469C for 45 s and 729C for 45 s for 3 cycles, and finally 959C for 30 s, 469 C for 45 s and 729C for 45 s for 45 cycles. After being purified, PCR products were cloned into the pGEM-T vector (Promega). Ten clones of each specimen were sequenced as described above. In vitro methylation of reporter constructs: The pCpGL-basic vector and pC-342/＋37 were treated with SssI methylase (New England Biolabs Japan, Inc., Tokyo, Japan) according to the manufacturer's instruction. In brief, 10–15 mg reporter constructs were incubated with SssI methylase (2 U/mg DNA) in the presence of 160 mM S-adenosylmethionine (New England Biolabs Japan) for 4 h at 379C. Unmethylated (control) reporter constructs were prepared as described above without SssI methylase. Control and methylated reporter constructs were then purified and used for reverse transfection. Com-

plete methylation of the pC-342/＋37 was confirmed by bisulfite sequencing as described above with specific primers, 5?-AAATTATTGATTTTTGTTTATGTGAGTAAA-3? (forward) and 5?-AAACCTTTCTTAATATTCTTAACATCCTC-3? (reverse).

Results Dependency of CYP1A2 promoter activity on the GC box located at －37/－32: To determine the promoter region of the CYP1A2 gene, we performed luciferase assay using five deletion constructs in HepG2 cells (Fig. 1A). Gradual deletion of the region －3188/ －343 from p4－3188/＋37 slightly decreased promoter activity, but p4－342/＋37 still retained activity comparable to that of p4－3188/＋37. When the region －342/－29, containing the GC box, was further deleted from p4－342/＋37, CYP1A2 promoter activity completely disappeared. To determine the role of the GC box in CYP1A2 promoter activity, we performed a mutation assay (Fig. 1B). Introduction of mutation in the GC box of p4－342/＋37 (p4－342/＋37mt) abolished promoter activity. Thus possibly, the GC box located at －37/－32 is an essential sequence for CYP1A2 promoter activity.

Fig. 1. Functional analysis of the GC box in the CYP1A2 promoter (A) Schematic representations of deletion constructs are shown on the left with their names. Black boxes indicate the GC box. Either one of the deletion constructs or a pGL4.17 vector and a pGL4.70 vector were transiently transfected into HepG2 cells for 48 h. The relative luciferase activity was calculated by normalizing to the renilla luciferase activity and is presented as a ratio to that of the control (pGL4.17). Each value is the mean±S.D. for three separate experiments, each performed in duplicate. (B) One of the reporter constructs (p4－342/＋ 37, p4－342/＋37mt or pGL4.17) and pGL4.70 were transfected into HepG2 cells for 48 h. Explanation of data normalization and value of luciferase assay is given above. (C) The reporter constructs (p4-PON1, p－342/＋37 and p4－342/＋37mt), pGL4.70 and expression vectors (Sp1 and Sp3 expression vectors) were transiently transfected into HepG2 (left) or into Drosophila SL2 cells (right) for 48 h. A p4-PON1 reporter construct was used as a positive control. Each value is the mean±S.D. of fold activation towards the activity obtained from the reporter promoter construct alone for three separate experiments, each performed in duplicate.

GC Box is Essential for Regulation of CYP1A2 Gene Expression

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Fig. 2. Association of CpG methylation status and CYP1A2 mRNA expression level (A) Location of CpG sites around the transcription start site is illustrated. The horizontal line indicates the region amplified by PCR from bisulfite-treated genomic DNA. The numbers above vertical lines indicate the position of the cytosine of CpG sites from the transcription start site. The black box indicates the GC box. TSS: transcription start site. (B) Expression profiles of CYP1A2 mRNA in human cell lines (left) and human tissue (right) were determined by RT-PCR analysis. PCR was performed three times independently, and representative data are shown. Li: liver, Lu: lung, K: kidney, SI: small intestine, N: negative control. (C) The region containing 4 CpG sites was amplified by PCR from bisulfite-treated genomic DNA extracted from human cell lines (left) and human tissue (right). PCR products were cloned into the plasmid and the clone was sequenced. Open circles () represent unmethylated cytosines and filled circles () represent methylated cytosines. Each row indicates an individual clone. The numbers in parentheses indicate methylation percentages at cytosine in the GC box.

To identify the transcription factors interacting with the GC box, we performed GMSA. The results showed that there were shifted bands thought to be Sp1 and Sp3, but no other shifted bands were detected (data not shown). Based on these findings, effects of Sp1 and Sp3 on CYP1A2 promoter activity were tested by a co-transfection assay in HepG2 and SL2 cells (Fig. 1C). In HepG2 cells, Sp1 and Sp3 stimulated luciferase activity of p4－342/＋37. However, the activity of p4－342/ ＋37mt was also stimulated by Sp1 and Sp3 to the same extent as that of p4－342/＋37. In SL2 cells, Sp1 and Sp3 did not stimulate luciferase activity of either p4－342/＋37 or p4－342/＋37mt. However, the level of luciferase activity of p4-PON1, a positive control, increased by co-transfection of Sp1 and Sp3 in both HepG2 and SL2 cells. These results suggest that Sp1 and Sp3 are not factors interacting with the GC box to activate CYP1A2 promoter, although they may bind to the GC box in vitro. Association between CpG methylation status in the GC box and CYP1A2 mRNA expression: We analyzed the sequence of the CYP1A2 promoter region –342/–29 to identify the CpG site and the only one was found in the GC box (Fig. 2A). We performed RT-PCR

analysis and bisulfite sequencing to examine the relationship between CYP1A2 mRNA expression profiles and methylation status of the GC box in human cell lines and human tissues (Figs. 2B and C). CYP1A2 mRNA expression was detected in a human hepatic cell line (HepG2 cells), but not in human extra-hepatic cell lines (Caco–2, HEK293 and HeLa cells) (Fig. 2B left). The methylation frequency of the GC box was 0% in HepG2 cells, while the frequencies were more than 80% in Caco-2, HEK293 and HeLa cells (Fig. 2C left). We also compared the methylation status of the GC box with CYP1A2 mRNA expression levels in human liver, lung, kidney and small intestine. CYP1A2 mRNA was detected in human liver but not in lung, kidney or small intestine (Fig. 2B right). The methylation frequencies of the GC box were 20% in human livers, while the frequencies were more than 80% in human lungs, kidneys and small intestines (Fig. 2C right). These results suggest that CpG methylation status of the GC box is significantly associated with CYP1A2 mRNA expression in human cell lines and human tissue. In addition to the GC box, there were three CpG sites located at ＋132/＋133, ＋168/＋169 and ＋203/ ＋204 in the region analyzed (Fig. 2A). The methylation frequency of the three CpG sites was more than 90% in

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Fig. 3. Effect of CpG methylation on CYP1A2 promoter activity pC－342/＋37 and pCpGL-basic were treated with Sss I methylase. Either the treated or untreated reporter construct and a pGL4.70 vector were transiently transfected into HepG2 cells for 48 h. Each value is the mean±S.D. of relative activities (firefly/renilla ) for three separate experiments, each performed in duplicate. Student's t -test was performed to determine significance of the difference between unmethylated and methylated reporter constructs. The symbol * indicates statistically significant differences compared with the unmethylated construct (pº0.05).

all human cell lines and human tissues except for human liver 2, in which the frequency of the CpG site located at ＋132/＋133 was 50% (Fig. 2C). Therefore, the methylation of these CpG sites did not appear associated with the expression of CYP1A2 mRNA in human cell lines and human tissues. Effects of CpG methylation of GC box on the CYP1A2 promoter activity: To determine the effects of GC box methylation on the promoter activity, we performed luciferase assays using methylated and unmethylated pC－342/＋37 in HepG2 cells. Luciferase activity of pCpGL-basic, which has no CpG site throughout the sequence, was not affected by SssI methylase treatment. The level of luciferase activity of methylated pC－342/＋37 was significantly (pº0.05) lower than that of unmethylated pC－342/＋37 (Fig. 3), although the difference was not so dramatic.

Discussion The results of the present study show that the GC box is an essential sequence for CYP1A2 promoter activity and that its methylation status is significantly associated with CYP1A2 mRNA expression in human cell lines and human tissue. Deletion assay showed that the region －342/－29 contained the minimal promoter in HepG2 cells (Fig. 1A). Although the existence of a GC box located at －37/－32 was reported by Chung et al.,4) its function in the CYP1A2 promoter remaines unclear. Our results show that CYP1A2 promoter activity is strongly dependent on the functional GC box (Fig. 1B), suggesting that the GC box is a master component of the CYP1A2 promoter. Narvaez et al.6) showed that the E-box and NF1-like element were also crucial for CYP1A2 promoter

activity, since introduction of mutation to the sites abolished promoter activity of the region －176/＋3. Therefore, the function of the GC box appears to be as important as that of the E-box and NF-1-like element in CYP1A2 promoter activity and all three cis-elements may cooperatively contribute to the activity. The lack of any of these elements may result in disassembly of the transactivating apparatus in the CYP1A2 promoter. Sp1 and/or Sp3 bind to the GC box in the promoter region to regulate gene expression in many cases. However, the results of cotransfection assay in SL2 cells indicate that neither Sp1 nor Sp3 is likely to be a transactivating factor interacting with the GC box in the CYP1A2 promoter (Fig. 1C). Nonetheless, Sp1 and Sp3 increased the level of promoter activity independent of the GC box in HepG2 cells. The reasons for this increase are unclear, but it is possible that Sp1 and Sp3 show coactivator-like behavior in this activation. It has been reported that Sp1 is recruited to a zinc-induced coactivator complex.20) Therefore, it may be an intriguing issue to address whether Sp1 and Sp3 stimulates CYP1A2 promoter through some factors expressed in HepG2 cells. To understand how the GC box enhances promoter activity is currently a challenge. However, there are two speculated roles of the GC box in the regulation of CYP1A2 promoter activity. First, some transcription factors, other than Sp1 and Sp3, could bind to the GC box to enhance CYP1A2 promoter activity. Kruppel-like factor 6 may be a candidate. It has been reported that this ubiquitously expressed factor binds to the GC box to enhance promoter activity of the urokinase-type plasminogen activator gene.21) Second, the GC box might be physically important for full promoter activity of the CYP1A2 gene. It has been suggested that flexibility of the promoter region is a critical factor affecting promoter activity.22) Su et al.23) showed that promoter activity of the rat bone sialoprotein gene was dependent on the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with the preinitiation complex on the TATA box. Taking into account that the GC box is located proximal to the TATA box in the CYP1A2 promoter, it is speculated that the GC box optimizes flexibility of the promoter region to align USF-1 and/or NF-1 with the preinitiation complex, leading to full promoter activity. Both mechanisms may simultaneously contribute to CYP1A2 promoter activity. Therefore, further studies are clearly required to clarify the mechanisms controlling CYP1A2 promoter activity by the GC box. Most recent studies examining the mechanism for tissue-specific regulation of gene expression by CpG methylation focus on CpG-rich promoters, containing CpG islands. However, it has been reported that chipmunk HP-27 gene expression is repressed by CpG methylation of the E-box in the promoter in extra-hepatic tissues,

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although the gene has a CpG-poor promoter.24) It has also been reported that expression of human lactogen I gene, which also has a CpG-poor promoter, is associated with the methylation status of the promoter.25) These findings suggest that CpG methylation in the CpG-poor promoter is significantly involved in the tissue-specific repression of gene expression. Given these reports, we postulate that methylation status of a single CpG site in the GC box in the CpG-poor promoter could be related to the expression status of the CYP1A2 gene. Our results clearly show that there is strong association between CpG methylation status in the GC box and CYP1A2 gene expression in human cell lines and human tissues (Figs. 2B and C). Therefore, the CpG site might be a targeted site of tissuespecific methylation, involved in the tissue-specific repression of CYP1A2 gene expression. Considering that CpG methylation in the GC box plays an important role in repression of CYP1A2 gene expression, how does methylation exert its role? In general, there are two mechanisms by which CpG methylation represses gene expression. First, CpG methylation directly inhibits transactivating factors from binding to ciselements.26,27) Second, methylated DNA recruits methylated-DNA binding proteins and histone deacetylases that alter chromatin modification and condensation. We could not determine whether the first mechanism is involved in expression of the CYP1A2 gene because the transcription factors binding to the GC box are not identified. Nevertheless, the contribution of the first mechanism appears less prominent, since the effect of CpG methylation in the GC box on CYP1A2 promoter activity was smaller than that of the introduction of mutation (Figs. 1B and 3). The second mechanism may be important for CYP1A2 gene repression. Chromatin modification and condensation might repress CYP1A2 gene expression by recruiting repressor complexes and/or by physically preventing transcription factors from binding to the cis-elements.28–30) Currently we do not have data on chromatin modification and condensation and therefore direct demonstration of the difference in chromatin status and DNA flexibility in the CYP1A2 promoter region in various tissues and cell lines would be an interesting challenge to explore the mechanism involved in the repression of CYP1A2 gene expression by the CpG methylation. In conclusion, our results suggest that a GC box located at －37/－32 of the CYP1A2 gene is a master component of the promoter, but the mechanism controlling CYP1A2 promoter activity by the GC box is unclear. Further studies are required to clarify whether some transcription factors and/or alteration of DNA flexibility is involved in this mechanism. Our results also suggest that the CpG site in the GC box is a targeted site of tissuespecific methylation, which may be involved in tissuespecific CYP1A2 gene expression. Since it is possible that

CpG methylation represses CYP1A2 gene expression by altering DNA flexibility with chromatin modification in the promoter region, further studies to determine the tertiary DNA structure and chromatin status of the CYP1A2 promoter region in the liver and other tissues are needed to understand the mechanism for the liverspecific expression of the CYP1A2 gene.

Acknowledgments: This work was supported in part by Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), MEXT, Japan. We thank Dr. Michael Rheli (Abteilung fur Hamatologie und Internistische Onkologie Klinikum der Universitat Regensburg, Germany) for providing pCpGLbasic vector. We thank Dr. Robert Tjian (Molecular and Cell Biology Department Howard Hughes Medical Institute U. C. Berkeley) for providing the pCMVSp1 plasmid, and also thank Dr. Guntarm Suske (Institut fuer Molekularbiologie und Tumorforschung (IMT) PhilippsUniversitaet, Marburg, Germany) for providing the pPacUsp3 plasmid. References 1)

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