Transcriptional Regulation of Rat Cyclin D1 Gene by CpG Methylation Status in Promoter Region
1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês
10.1074/jbc.274.40.28787
ISSN1083-351X
AutoresSohei Kitazawa, Riko Kitazawa, Sakan Maeda,
Tópico(s)Cancer-related Molecular Pathways
ResumoCyclin D1, a G1/S cell cycle-regulating oncogene, is known to be transcriptionally regulated by numerous growth factors. We cloned and characterized the rat cyclin D1 gene 5′-flanking region and, by species- and subspecies-matched transient transfection studies, found that a basic promoter structure with a cAMP response element and two continuous Sp1-binding sites was crucial for the steady-state expression of the cyclin D1 gene. Furthermore, the methylation status especially around two continuous Sp1-binding sites was found to be an important epigenetical mechanism determining the steady-state expression level in rat leukemic cell lines K4D, K4DT, and K4D16. Whether or not epigenetic control of the cyclin D1 gene existed among normal rat tissues was further examined by high sensitivity mapping of the methylated cytosine. In normal rat tissues, the methylated cytosines at non-CpG loci within two continuous Sp1-binding sites were observed in uterine stromal cells of the basal layer and found to be demethylated in the functioning layer, possibly by a passive demethylation mechanism through cell division. Since in the passive demethylation process Sp1-binding sites remain methylated in a part of the cell population, methylated cytosines at Sp1-binding sites may be essential for keeping a number of the stromal cells in the basal layer live against estrogen-induced proliferation that leads to either apoptosis or compaction. Cyclin D1, a G1/S cell cycle-regulating oncogene, is known to be transcriptionally regulated by numerous growth factors. We cloned and characterized the rat cyclin D1 gene 5′-flanking region and, by species- and subspecies-matched transient transfection studies, found that a basic promoter structure with a cAMP response element and two continuous Sp1-binding sites was crucial for the steady-state expression of the cyclin D1 gene. Furthermore, the methylation status especially around two continuous Sp1-binding sites was found to be an important epigenetical mechanism determining the steady-state expression level in rat leukemic cell lines K4D, K4DT, and K4D16. Whether or not epigenetic control of the cyclin D1 gene existed among normal rat tissues was further examined by high sensitivity mapping of the methylated cytosine. In normal rat tissues, the methylated cytosines at non-CpG loci within two continuous Sp1-binding sites were observed in uterine stromal cells of the basal layer and found to be demethylated in the functioning layer, possibly by a passive demethylation mechanism through cell division. Since in the passive demethylation process Sp1-binding sites remain methylated in a part of the cell population, methylated cytosines at Sp1-binding sites may be essential for keeping a number of the stromal cells in the basal layer live against estrogen-induced proliferation that leads to either apoptosis or compaction. Among cyclins, expression of the D types cyclins is the earliest event in G1 phase that leads to cell division (1Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1993) Google Scholar, 2Quelle D.E. Ashumn R.A. Shurtleff S.A. Kato J. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (979) Google Scholar, 3Jiang W. Kahan N. Tomita N. Zhang Y. Lu S. Weinstein I.B. Cancer Res. 1992; 52: 2980-2983PubMed Google Scholar). Cyclin D1, first isolated from murine bone marrow macrophages as one of the early responsive genes after the stimulation of colony stimulating factor 1 (M-colony stimulating factor) (4Matusshime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (990) Google Scholar), together with cyclin-dependent kinase (cdk)-4 and -6, regulates the G1/S cell cycle through inactivation of the retinoblastoma protein by phosphorylation (1Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1993) Google Scholar, 5Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar, 6Dowdy S.F. Hinds P.W. Louie K. Reed S.I. Arnold A. Weinberg R.A. Cell. 1993; 73: 499-511Abstract Full Text PDF PubMed Scopus (691) Google Scholar, 7Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2935) Google Scholar). Besides colony stimulating factor-1, cyclin D1 is known to be transcriptionally up-regulated by numerous growth factors like the nerve growth factor in PC12 cells (8Yan G-Z. Ziff E.B. J. Neurosci. 1997; 17: 6122-6132Crossref PubMed Google Scholar), estrogen, and gestergen in endometrial cells (9Prall O.W.J. Saecevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) and the epidermal growth factor in prostate cancer cells (10Perry J.E. Grossmann M.E. Tindall D.J. Prostate. 1998; 35: 117-124Crossref PubMed Scopus (89) Google Scholar). To investigate the transcription regulating mechanism of cyclins, promoter structures of the cyclins have been studied (11Herber B. Truss M. Beato M. Muller R. Oncogene. 1994; 9: 1295-1304PubMed Google Scholar, 12Motokura T. Arnold A. Genes Chromosomes & Cancer. 1993; 7 (65): 89Crossref PubMed Scopus (146) Google Scholar, 13Yan Y-X. Nakagawa H. Lee M-H. 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The basic promoter structure of the 5′-regulatory region of the cyclin D1 gene has been demonstrated as a TATA-less promoter with a CRE 1The abbreviations used are:CREcAMP response elementCREBcAMP response element-binding proteinPCRpolymerase chain reactionPIPES1,4-piperazinediethanesulfonic acid and two continuous Sp1-binding sites through which most of the growth factors exert their cell proliferative stimuli (8Yan G-Z. Ziff E.B. J. Neurosci. 1997; 17: 6122-6132Crossref PubMed Google Scholar, 11Herber B. Truss M. Beato M. Muller R. Oncogene. 1994; 9: 1295-1304PubMed Google Scholar, 12Motokura T. Arnold A. Genes Chromosomes & Cancer. 1993; 7 (65): 89Crossref PubMed Scopus (146) Google Scholar, 13Yan Y-X. Nakagawa H. Lee M-H. Rustgi A.K. J. Biol. Chem. 1997; 272: 33181-33190Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Promoter structures of the cyclin D2 and D3 genes are also TATA-less with a number of Sp1-, AP1-, and AP2-binding sites (14Brooks A. Shiffman D. Chan C.S. Brooks E.E. Milner P.G. J. Biol. Chem. 1996; 271: 9090-9099Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), indicating that these three D-type cyclins share similar cell cycle-dependent regulating mechanisms with complex mutuality. cAMP response element cAMP response element-binding protein polymerase chain reaction 1,4-piperazinediethanesulfonic acid On the other hand, aberrant methylation of CpG loci within 5′-regulatory regions play a principal role in the tissue-specific expression of genes by affecting the interactions of DNA with chromatin proteins and transcription factors (17Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1083) Google Scholar, 18Tate P. Skarnes W. Bird A. Nat. Genet. 1996; 12: 205-208Crossref PubMed Scopus (187) Google Scholar, 19Simmen M.W. Leitgeb S. Charlton J. Jones S.J. Harris B.R. Clark V.H. Bird A. Science. 1999; 283: 1164-1167Crossref PubMed Scopus (118) Google Scholar, 20Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. Baylin S.B. Nat. Genet. 1999; 21: 103-107Crossref PubMed Scopus (1680) Google Scholar). In cell cycle-regulating genes, cyclin D2 (21Sinclair A.J. Palmero I. Holder A. Peters G. Farrell P.J. J. Virol. 1995; 69: 1292-1295Crossref PubMed Google Scholar) and two kinds of INK4 class cyclin-dependent kinase inhibitors, p16ink4b and p15ink4b (22Mao L. Merlo A. Bedi G. Shapiro G.I. Edwards C.D. Rollins B.J. Sidransky D. Cancer Res. 1995; 55: 2995-2997PubMed Google Scholar, 23Herman J.G. Jen J. Merlo A. Baylin S.B. Cancer Res. 1996; 56: 722-727PubMed Google Scholar, 24Herman J.G. Civin C.I. Issa J.P. Collector M.I. Sharkis S.J. Baylin S.B. Cancer Res. 1997; 57: 837-841PubMed Google Scholar), are regulated by CpG methylation, and de novohypermethylation of the 5′-CpG island of p16ink4b and p15ink4b is common in malignant tumors (22Mao L. Merlo A. Bedi G. Shapiro G.I. Edwards C.D. Rollins B.J. Sidransky D. Cancer Res. 1995; 55: 2995-2997PubMed Google Scholar, 23Herman J.G. Jen J. Merlo A. Baylin S.B. Cancer Res. 1996; 56: 722-727PubMed Google Scholar, 24Herman J.G. Civin C.I. Issa J.P. Collector M.I. Sharkis S.J. Baylin S.B. Cancer Res. 1997; 57: 837-841PubMed Google Scholar, 25Villuendas R. Sanchez-Beato M. Martinez J.C. Saez A.I. Martinez-Delgado B. Garcia J.F. Mateo M.S. Sanchez-Verde L. Benitez J. Martinez P. Piris M.A. Am. J. Pathol. 1998; 153: 887-897Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), indicating that the function of these genes may be epigenetically lost during tumor progression (25Villuendas R. Sanchez-Beato M. Martinez J.C. Saez A.I. Martinez-Delgado B. Garcia J.F. Mateo M.S. Sanchez-Verde L. Benitez J. Martinez P. Piris M.A. Am. J. Pathol. 1998; 153: 887-897Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The mechanisms of gene silencing by methylated cytosine are, however, varied among promoters (26Chomet P.S. Curr. Opin. Cell Biol. 1991; 3: 438-443Crossref PubMed Scopus (26) Google Scholar). The most generally reported mechanism is repression of transcription by methyl CpG-binding proteins (MeCP1 and MeCP2) that bind DNA in a sequence independent manner: binding of methyl-CpG-binding proteins results in alternating the chromatin structure and preventing the transcriptional factors like Sp1 from DNA binding (17Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1083) Google Scholar, 18Tate P. Skarnes W. Bird A. Nat. Genet. 1996; 12: 205-208Crossref PubMed Scopus (187) Google Scholar, 19Simmen M.W. Leitgeb S. Charlton J. Jones S.J. Harris B.R. Clark V.H. Bird A. Science. 1999; 283: 1164-1167Crossref PubMed Scopus (118) Google Scholar). To analyze the mechanism of overexpression of the cyclin D1 gene in leukemic cell lines, we first isolated and characterized the 5′-regulatory region of Long-Evans rat cyclin D1 gene and then assessed the possibility of epigenetic control of gene expression by aberrant cytosine methylation of the CpG dinucleotide. Whether or not such epigenetic control of the cyclin D1 gene existed naturally among normal rat tissues was further examined in microdissected samples by the recently introduced high sensitivity mapping of methylated cytosine (27Olek A. Oswald J. Walter J. Nucleic Acids Res. 1996; 24: 5064-5066Crossref PubMed Scopus (336) Google Scholar). We found that the steady-state expression of the cyclin D1 gene was influenced by the methylation status, as an epigenetic event, of its 5′-flanking region in rat leukemic and endometrial stromal cells. Long-Evans rat-derived 7,12-dimethylbenz[a]anthracene-induced leukemic cell lines, K2D, K3D, K4D, K4D16, and K4DT, established in our laboratory (28Maeda S. Uenaka H. Ueda N. Shiraishi N. Suglyama T. J. Natl. Cancer Inst. 1980; 64: 539-546PubMed Google Scholar), were cultured and maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% fetal bovine serum (Sigma). Original K2D, K3D, and K4D show non-adherent growth to the plastic culture dish. K4DT (29Fujita M. Takahashi R. Kitada K. Watanabe R. Kitazawa S. Ashoori F. Liang P. Saya H. Serikawa T. Maeda S. Cancer Lett. 1997; 112: 47-55Crossref PubMed Scopus (11) Google Scholar) and K4D16 from K4D were subcloned by Hamberger and Salmon's double-layered soft agarose method (30Hamburger A.W. Salmon S.E. Science. 1977; 197: 461-463Crossref PubMed Scopus (1716) Google Scholar). These two cell lines show monocyte/macrophage phenotypes, adherent growth to the plastic culture dish, latex-phagocytosis, positively for α-naphthyl butyrate esterase, and negativity for benzidine staining (29Fujita M. Takahashi R. Kitada K. Watanabe R. Kitazawa S. Ashoori F. Liang P. Saya H. Serikawa T. Maeda S. Cancer Lett. 1997; 112: 47-55Crossref PubMed Scopus (11) Google Scholar). Total cellular RNA was extracted from rat leukemic cell lines K2D, K3D, K4D, K4D16, and K4DT by RNAzol (Tel-Test, Inc., Friendswood, TX). RNA samples (10 μg) were separated by denaturing electrophoresis in formaldehyde-agarose gels, and stained with ethidium bromide. The RNA was transferred onto Hybond N+ nylon membranes (Amersham Pharmacia Biotech) and immobilized by UV cross-linking, prehybridized and hybridized to32P-labeled cDNA probes in 50% formamide, 6 × SSC, 10 × Denhardt's solution, 10 mm EDTA, 0.1% SDS, and 150 μg/ml denatured salmon sperm DNA at 42 °C for 16 h. The membranes were washed twice in 2 × SSC containing 0.1% SDS, 1 × SSC containing 0.1% SDS and finally 0.1 × SSC containing 0.1% SDS at 60 °C and then analyzed with image analyzer BAS-EWS 4075 (FUJIX, Tokyo, Japan). The rat cyclin D1 cDNA probe (31Kitazawa S. Ross F.P. McHugh K. Teitelbaum S.L. J. Biol. Chem. 1994; 270: 4115-4200Abstract Full Text Full Text PDF Scopus (55) Google Scholar) was a kind gift from Dr. H. Okayama (Department of Molecular Genetics, Osaka University, Japan). The relative expression level was estimated by the optical density of the cyclin D1 mRNA band standardized by that of rat glyceraldehyde-6-phosphate dehydrogenase. Subconfluent cells in 100-mm culture dishes were lysed with 1.0 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.2% Nonidet P-40, 0.2% sodium deoxycholate, 0.1% SDS, and 50 μm/ml aprotinin). After sonication for 1 min, lysates were centrifuged at 15,000 rpm for 20 min. Supernatants, equalized by protein concentration, were separated by SDS-polyacrylamide gel electrophoresis, transferred to the nylon membrane (Amersham), and immunoblotted with a primary antibody against human, rat, and mouse cyclin D1 (R-124, Santa Cruz Biotechnology). Immunocomplexes were visualized using horseradish peroxidase-conjugated secondary antibodies with cobalt and nickel intensifiers. Cells were washed twice with ice-cold phosphate-buffered saline and collected by scrapping in 1 × SSC before being centrifuged at 1500 rpm for 5 min. Nuclei were extracted in lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mmNaCl, 3 mm MgCl2, 0.5% Nonidet P-40) for 10 min on ice, and centrifuged at 3000 rpm for 5 min. The pellets were washed twice with lysis buffer and resuspended with 0.5 ml of suspension buffer (50 mm Tris-HCl, pH 8.3, 40% glycerol, 5 mm MgCl2, 0.1 mm EDTA). The suspended nuclei were mixed with 100 μl of reaction mixture (10 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 300 mm KCl, 0.5 mm each of ATP, CTP, GTP, 100 μCi of [α-32P]UTP 3000 μCi/mm), and transcribed, in vitro, for 30 min at 30 °C (31Kitazawa S. Ross F.P. McHugh K. Teitelbaum S.L. J. Biol. Chem. 1994; 270: 4115-4200Abstract Full Text Full Text PDF Scopus (55) Google Scholar). After DNase I treatment (final concentration 20 μg/ml) for 30 min at 30 °C, transcription was halted with 200 μl of stop solution (20 mm Tris-HCl, pH 7.4, 2% SDS, 10 mm EDTA, 200 μg/ml proteinase K), and the nuclei were incubated for 30 min at 42 °C. After extraction in phenol/chloroform, 50 μg of carrier RNA and 5 ml of ice-cold 5% trichloroacetic acid were added to the supernatant; the solution was incubated for 30 min on ice and then filtered through a nitrocellulose membrane (2.5 cm in diameter, Amersham Pharmacia Biotech). The blotted membrane was washed three times with 3% trichloroacetic acid and incubated with 0.9 ml of the incubation solution (20 mm HEPES, pH 7.5, 5 mmMgCl2, 1 mm CaCl2, 25 μg/ml DNase I) for 30 min at 37 °C. Labeled RNA was eluted by incubation in 30 μl of 0.5 m EDTA and 100 μl of 10% SDS for 10 min at 60 °C, purified by proteinase K (25 μg/ml) treatment for 30 min at 37 °C and ethanol precipitation, and finally dissolved in 50 μl of 10 mm Tris-HCl, pH 7.5 and 2 mm EDTA. A quantity of 10 μg each of rat cyclin D1 cDNA, rat glyceraldehyde 3-phosphate dehydrogenase cDNA, and vector DNA was denatured in 50 μl of 0.2 n NaOH for 30 min at 27 °C, neutralized with 500 μl of 6 × SSC, slot-blotted onto nylon membranes saturated with H2O and 6 × SSC, and probed with transcribed RNA in hybridization solution (50% formamide, 5 × SSC, 50 mm sodium phosphate, pH 6.5, 1 × Denhardt's solution, 1 × Background Quencher (Tel-Test, Inc.)) mixed at 50% (v/v) with dextran sulfate for 48 h at 42 °C. The membranes were washed with 2 × SSC, 0.1% SDS for 15 min at 27 °C, and with 0.2 × SSC, 0.1% SDS for 30 min at 65 °C, and exposed to Kodak X-Omat film for 2 days at −80 °C. High-molecular weight genomic DNA was isolated and purified from the Long-Evans rat leukemic cell line, K4D, by standard procedures. A quantity of 10 μg of rat genomic DNA was completely digested with BglII then ligated into the BamHI site of the EMBL3 phage vector (Promega), and packaged with GigaPack gold kit (Stratagene). The original Long-Evans genomic DNA library contained 5 × 106 independent phage plaques with an average insert size of 11.0 kilobase pairs. The rat genomic library was screened with a PCR amplified rat cyclin D1 (32Tamura K. Kanaoka Y. Jinno S. Nagata A. Ogiso Y. Shimizu K. Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar) exon I partial cDNA fragment. After tertial screening, theSacI-digested portion of the genomic clone was subcloned into the plasmid pGEM 7Z(+) (Promega), and sequenced by the ideoxynucleotide termination method. Primer extension was carried out as described previously (33Kitazawa S. Kitazawa R. Tamada H. Maeda S. Biochim. Biophys Acta. 1998; 1443: 358-363Crossref PubMed Scopus (22) Google Scholar). Briefly, 100 ng of the oligonucleotide, complementary to nucleotides of the cyclin D1 cDNA, was labeled with T4 polynucleotide kinase to a specific activity of 2 × 108 cpm/g. A 2.0 × 106 cpm of the oligonucleotide was annealed for 2 h at 40 °C in 80% formamide, 1 mm EDTA, 400 mm NaCl, and 50 mm PIPES, pH 6.4, to 5 μg of poly(A)+mRNA from K4DT in the presence of avian myeloblastosis virus reverse transcriptase (Promega). The extended cDNA was analyzed by denaturing sequencing gel with sequencing reaction products as a marker. The putative promoter region was ligated into a reporter gene vector, pGL-3luc (Promega), atXbaI-SacI sites. A series of deletion constructs was generated either by restriction endonuclease digestion or PCR amplification of the desired portion of the insert. The minimal cyclin D1 promoter-reporter gene construct (−105), containing 4 CpG loci, was methylated in vitro by SssI (CpG) methylase (Takara, Kyoto, Japan) with 5 mm S-adenosylmethionine. Purified promoter-reporter gene constructs were transiently transfected into rat leukemia cell lines K4D, K4D16, and K4DT with Tfx™-50 reagent at a charge ratio of 1:4 according to the manufacturer's instructions (Promega). Tissue samples from Long-Evans rats were fixed with 4% paraformaldehyde and embedded in paraffin; 4 μm-thick sections were then cut and dewaxed through xylene and a series of graded alcohols. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min. Specimens were then incubated with 2% non-fat dry milk in phosphate-buffered saline for 10 min and primary antibody against human, rat, and mouse cyclin D1 (R-124, Santa Cruz Biotechnology) for 30 min. After three 10-min washing with phosphate-buffered saline, specimens were incubated with rabbit anti-mouse IgG preabsorbed with non-immunized rat serum for 30 min. Finally, the cyclin D1 protein was immunolocalized by the streptavidine-biotin peroxidase complex method. Each genomic DNA, extracted and purified from normal rat fetus, various organs from adult rats, K4D, K4DT, and K4D16 cell lines, was digested with either HapII or MspI restriction enzymes and electrophoresed on 1.2% agarose gels. After transferring onto nylon membranes (Amersham Pharmacia Biotech), blotted DNA was probed with 1.4 kilobases of the 5′-flanking region of the Long-Evans rat cyclin D1 gene. After washing twice in 2 × SSC containing 0.1% SDS, 1 × SSC containing 0.1% SDS and finally 0.1 × SSC containing 0.1% SDS at 60 °C, the membranes were analyzed with image analyzer BAS-EWS 4075 (FUJIX). The bisulfite reaction was carried out according to the procedures of Frommer et al. (34Frommer M. McDonald L.E. Millar D.S. Collis C.M. Watt F. Grigg G.W. Molloy P.L. Paul C.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1827-1831Crossref PubMed Scopus (2525) Google Scholar) and Olek et al. (27Olek A. Oswald J. Walter J. Nucleic Acids Res. 1996; 24: 5064-5066Crossref PubMed Scopus (336) Google Scholar). A quantity of 1 μg of DNA in a volume of 50 μl of TE was denatured by NaOH (final concentration, 0.2 m) for 10 min at 37 °C. Freshly prepared 30 μl of 10 mm hydroquinine and 520 μl of 3 m sodium bisulfite at pH 5 were added to the samples. Each sample was incubated under mineral oil at 50 °C for 16 h. Modified DNA was purified with Wizard DNA purification resin according to the manufacturer's recommended protocol (Promega) and eluted into 50 μl of H2O. Modification was completed by NaOH (final concentration, 0.3 m) treatment for 5 min at room temperature, then by ethanol precipitation. DNA was resuspended in 20 μl of H2O and used immediately or stored at −20 °C. Paraffin-embedded samples were deparaffinized in xylene and buried in agarose bead and then treated with Pronase K at 50 °C for 12 h according to the method described. Bisulfite-modified DNA (100 ng) was amplified with nested PCR using the following sets of primers, 5′-GTGTTGATGAAATTGAAAGAAGTTG-3′, sense; 5′-ACTTTACAACTTCAACAAAACTCCCCTAT-3′, antisense. Each primer sequence was set not to contain the CpG loci of the rat cyclin D1 5′-flanking region. The PCR condition was as follows: 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and the final elongation step for 5 min at 72 °C. The PCR mixture contained 1 × buffer (Takara) with 1.5 mm MgCl2, 20 pmol of each primer, 0.2 mm dNTPs, and bisulfite-modified DNA (50 ng) in a final volume of 50 μl. Each PCR product was loaded onto 3% agarose gels, stained with ethidium bromide, and visualized under UV illumination; the fragments were subjected to automated DNA sequencing. The sequencing reactions for the products of the PCR were carried out with a DNA sequencing kit (Perkin-Elmer) by the dideoxy nucleotide termination method using the PCR condition. Reaction products were analyzed on a 310 Genetic Analyzer (Perkin-Elmer). Among the rat leukemic cell lines, K4D16 and K4DT cells with adherence to the culture dish overexpressed the cyclin D1 gene as demonstrated by Northern blot analysis (Fig. 1 A). To analyze whether or not the cyclin D1 overexpression at the mRNA level related to its translation, cyclin D1 protein expression was checked by Western blotting; mirroring Northern blot analysis, K4D16 and K4DT expressed 5 times as much cyclin D1 protein as the original strain, K4D (Fig. 1 B). To analyze the mechanism determining the difference in the steady-state gene expression of cyclin D1 between the original K4D monocytic cell line and the subcloned K4D16 and K4DT cells, the rate of transcription in these cell lines was examined by nuclear run-on assay; K4D16 and K4DT cells transcribed 5 times as much cyclin D1 mRNA as that of K2D, K3D, and K4D while keeping the basic transcription rate for glyceraldehyde-6-phosphate dehydrogenase constant (Fig. 1 C). These results suggested that K4D16 and K4DT cells overexpressed cyclin D1 mainly at the transcriptional level. Two positive clones obtained after tertial screening were identical by the restriction map of the Southern blot analysis (data not shown). Restriction mapping of the genomic insert is shown in Fig. 3 A. A 2.4-kilobaseSacI-SacI portion of the subcloned insert contained the whole 5′-untranslated portion and part of the exon 1 region of the published rat cyclin D1 cDNA (32Tamura K. Kanaoka Y. Jinno S. Nagata A. Ogiso Y. Shimizu K. Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar). As shown in Fig.2, a reverse transcriptase-mediated extension of the antisense oligonucleotide primer to poly(A)+ mRNA from K4D16 yielded one major and three minor extended fragments, positioning the major transcriptional start site 99 nucleotides upstream from the initial methionine site; this was assigned the +1 position. Fig.3 B shows the part of the sequence with putative binding domains. The 5′-flanking sequence around the site of transcription initiation showed that the cyclin D1 gene lacked the canonical TATA box, but did contain two continuous Sp1-binding sites at −109 and one CRE site at −48. An additional Sp1-binding site, one octamer sequence, an E-box, and E2F-binding sites were located at −476, −209, −532, and −679, respectively. To assess the promoter activity and cis-regulatory elements, a series of deletion constructs of the 5′-flanking sequence were ligated into the PGL-3 vector. As shown in Fig. 4, unlike Northern and Western blots and nuclear run-on analysis, K4D, K4D16, and K4DT cells showed almost the same level of luciferase activity for each construct, and a 105-base pair fragment with a single Sp1 site had basic promoter activity. Methylation of minimal promoter construct (Met-105) at CpG sites by SssI, however, diminished that promoter activity to 32–36% in the three leukemic cell lines, suggesting that the difference of the transcription rate of the cyclin D1 gene was mainly an epigenetical event. On the other hand, the weak negatively regulating element was located between −825 and −615.Figure 2Rat cyclin D1 genomic sequence and mapping the sites of transcriptional initiation. Reverse transcriptase-mediated extension of the antisense oligonucleotide primer to poly(A)+ mRNA from K4D16 leukemic cell lines yielded one major (large arrow with +1) and three minor extended fragments (small arrows), positioning the major transcriptional start site 99 nucleotides upstream from the initial methionine site (assigned the +1 position).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Transient transfection study with a series of deletion constructs. A series of the deletion constructs were made by either restriction enzymatic digestion or PCR amplification. Unlike Northern and Western blots and nuclear run-on analysis, K4D, K4D16, and K4DT cells showed almost same level of the luciferase activity for each construct, and a 105-base pair fragment with a single Sp1 site had basic promoter activity. In the steady-state, deletion of the −825 to −615 portion increased luciferase activity by 30%, indicating the presence of a weak negatively regulating element within that portion. When minimal promoter construct (−105) was methylated bySssI at CpG sites (Met-105), luciferase activity decreased to 32–36% in all leukemic cell lines, suggesting that the difference in the transcription rate of the cyclin D1 was mainly a epigenetical event.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Fig. 6 A, since in the 5′-flanking region of the rat cyclin D1 gene numerous CpG loci constituted a CpG island, where methylation often contributes one of the major cis-regulating elements, we further tested whether or not the transcriptional activity of the cyclin D1 gene was related to the methylation status of the CpG loci. The methylation status of the 5′-flanking region of the rat cyclin D1 gene was first surveyed by Southern blot analysis. As shown in Fig.5, HapII digestion of genomic DNA from K4D showed methylation-protected bands. Furthermore,HapII-protected bands observed in K4D were demethylated in genomic DNA from K4D16 and K4DT cells. In genomic DNA extracted from normal adult Long-Evans rats, minor HapII-protected bands were also observed in ovary and uterus (Fig. 5, arrows). To access more precise information about the methylation status including non-CpG methylated cytosine in leukemic cell lines and normal tissues, high sensitivity mapping of the methylated cytosine was carried out by sodium bisulfate modification before PCR amplification. As expected, genomic Southern blot analysis revealed that some of the methylated cytosines found in the K4D cell line were demethylated in K4D16 and K4DT cells; especially, a methylated cytosine at the CpG locus located between the two continuous Sp1-binding sites (5 in Fig.6 B) in K4D cell line was demethylated in the K4D16 and the K4DT cell lines (Fig. 6 C). Among normal rat tissues, however, the occurrence of methylated cytosines was less frequent than among rat leukemic cell lines, and partial methylation of the cytosine at non-CpG loci around two continuous Sp1-binding sites (asterisk in Fig. 6,A and B) was noted in the ovary and uterus. These results suggested that methylation of the 5′-regulatory region of the rat cyclin D1 gene was manifested not merely as an in vitroartificial event but as a
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