JBC Focus on PI3-Kinase with Echelon

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kitazawa, S.
Right arrow Articles by Maeda, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kitazawa, S.
Right arrow Articles by Maeda, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 40, 28787-28793, October 1, 1999


Transcriptional Regulation of Rat Cyclin D1 Gene by CpG Methylation Status in Promoter Region*

Sohei KitazawaDagger , Riko Kitazawa, and Sakan Maeda

From the Second Department of Pathology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Among cyclins, expression of the D types cyclins is the earliest event in G1 phase that leads to cell division (1-3). 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) (4), together with cyclin-dependent kinase (cdk)-4 and -6, regulates the G1/S cell cycle through inactivation of the retinoblastoma protein by phosphorylation (1, 5-7). 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 (8), estrogen, and gestergen in endometrial cells (9) and the epidermal growth factor in prostate cancer cells (10). To investigate the transcription regulating mechanism of cyclins, promoter structures of the cyclins have been studied (11-16). The basic promoter structure of the 5'-regulatory region of the cyclin D1 gene has been demonstrated as a TATA-less promoter with a CRE1 and two continuous Sp1-binding sites through which most of the growth factors exert their cell proliferative stimuli (8, 11-13). Promoter structures of the cyclin D2 and D3 genes are also TATA-less with a number of Sp1-, AP1-, and AP2-binding sites (14), indicating that these three D-type cyclins share similar cell cycle-dependent regulating mechanisms with complex mutuality.

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 (17-20). In cell cycle-regulating genes, cyclin D2 (21) and two kinds of INK4 class cyclin-dependent kinase inhibitors, p16ink4b and p15ink4b (22-24), are regulated by CpG methylation, and de novo hypermethylation of the 5'-CpG island of p16ink4b and p15ink4b is common in malignant tumors (22-25), indicating that the function of these genes may be epigenetically lost during tumor progression (25). The mechanisms of gene silencing by methylated cytosine are, however, varied among promoters (26). 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 (17-19).

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 (27). 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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Long-Evans rat-derived 7,12-dimethylbenz[a]anthracene-induced leukemic cell lines, K2D, K3D, K4D, K4D16, and K4DT, established in our laboratory (28), 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 (29) and K4D16 from K4D were subcloned by Hamberger and Salmon's double-layered soft agarose method (30). These two cell lines show monocyte/macrophage phenotypes, adherent growth to the plastic culture dish, latex-phagocytosis, positively for alpha -naphthyl butyrate esterase, and negativity for benzidine staining (29).

Northern Blot Analysis-- 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 to 32P-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 (31) 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.

Western Immunoblotting-- 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.

Nuclear Run-on Assay-- 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 mM NaCl, 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 [alpha -32P]UTP 3000 µCi/mM), and transcribed, in vitro, for 30 min at 30 °C (31). 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 mM MgCl2, 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.

Isolation, Subcloning, and Sequencing of Rat Cyclin D1 Genomic Clones-- 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 (32) exon I partial cDNA fragment. After tertial screening, the SacI-digested portion of the genomic clone was subcloned into the plasmid pGEM 7Z(+) (Promega), and sequenced by the ideoxynucleotide termination method.

Mapping the Site of Cyclin D1 Transcriptional Initiation-- Primer extension was carried out as described previously (33). 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.

Expression, Plasmid Construction, Transient Transfections, and Reporter Gene Assays-- The putative promoter region was ligated into a reporter gene vector, pGL-3luc (Promega), at XbaI-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 TfxTM-50 reagent at a charge ratio of 1:4 according to the manufacturer's instructions (Promega).

Immunohistochemistry-- 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.

Mapping of Methylation Status by Southern Blot Screening and Sodium Bisulfite Modification-- 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. (34) and Olek et al. (27). 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Northern, Western Blot, and Nuclear Run-on Analysis of Cyclin D1 Genes in a Steady-state Condition-- 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. 1A). 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. 1B). 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. 1C). These results suggested that K4D16 and K4DT cells overexpressed cyclin D1 mainly at the transcriptional level.


View larger version (44K):
[in this window]
[in a new window]
  		Fig. 1.   Northern, Western blot, and nuclear run-on analysis of cyclin D1 genes in a steady-state condition of rat leukemic cell lines. A, equal amounts of total RNA extracted from rat leukemic cell lines, K2D, K3D, K4D, K4D16, and K4DT were electrophoresed and probed with 32P-labeled rat cyclin D1 cDNA. At the steady-state level, K4D16 and K4DT cells overexpressed cyclin D1 gene 5 times as much as the other leukemic cell lines. B, cell lysates extracted from each cell line were electrophoresed and probed with anti-cyclin D1 protein antibody; mirroring the Northern blot analysis, K4D16 and K4DT expressed 5 times as much cyclin D1 protein as the original strain, K4D. C, the rate of transcription in the cell lines was assessed 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 (G6PDH) constant.

Rat Cyclin D1 Genomic Sequence and Mapping the Sites of Transcriptional Initiation-- 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. 3A. A 2.4-kilobase SacI-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 (32). 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. 3B 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.


View larger version (76K):
[in this window]
[in a new window]
  		Fig. 2.   Rat 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 larger version (56K):
[in this window]
[in a new window]
  		Fig. 3.   Restriction map and sequences of the genomic insert. A, restriction map of the cloned insert. B, part of the sequence with putative binding domains. The 5'-flanking sequence around the site of transcription initiation shows that the cyclin D1 gene lacks the canonical TATA box, but does contain two continuous Sp1-binding sites at -109 and one CRE site at -48. An additional Sp1-binding site, an octamer sequence, an E-box, and a putative E2F-binding site are located at -476, -209, -532, and -679, respectively. Four transcriptional start sites are indicated by arrows.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4.   Transient 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 by SssI 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.

Mapping Methylation Status by Southern Blot Screening and Sodium Bisulfite Modification-- As shown in Fig. 6A, 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. 6B) in K4D cell line was demethylated in the K4D16 and the K4DT cell lines (Fig. 6C). 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 vitro artificial event but as an instinctive event at least in these tissues. To identify the cells bearing methylated cytosines, the normal rat uterus was microdissected from 7-µm thick paraffin sections into the major histological components, endometrial stromal cells from the functional layer (Fig. 7B, 1 and 2), the basal layer (Fig. 7B, 3 and 4), and the methylated cytosine was analyzed by the agarose-bead mediated high sensitive mapping technique (27). To compare the methylation status of cyclin D1 promoter and the expression of the cyclin D1 protein, serial tissue sections were immunohistochemically stained for cyclin D1. As shown in Fig. 7C, 3 and 4, dissected components of stromal cells in the basal layer (Fig. 7B, 3 and 4) showed methylated cytosines at non-CpG loci in the first Sp1-binding site. On the other hand, dissected components of proliferating stromal cells in the functional layer (Fig. 7B, 1 and 2) showed either hemi-methylated or non-methylated cytosines (Fig. 7C, 1 and 2). Heterogeneity of the site of the methylated cytosines in endometrial stromal cells was also noted. Reflecting the methylation status of the Sp1 sites, immunohistochemistry revealed cyclin D1 protein exclusively among the cells in the functional layer (Fig. 7B), where no methylation was observed at the Sp1-binding sites.


View larger version (104K):
[in this window]
[in a new window]
  		Fig. 5.   Southern blot analysis by methylation resistant MspI and methylation sensitive HapII isoschizomer. Genomic DNA was extracted and purified from various normal tissues from 24-week-old Long-Evans rats, and digested either by MspI (M) or HapII (H) before electrophoresis. Southern blot analysis was carried out using Long-Evans rat cyclin D1 gene 5'-flanking fragment as a probe. By HapII digestion genomic DNA from K4D showed heavy methylation by protected bands, which were in part demethylated in K4D16 cells. In genomic DNA extracted from normal adult Long-Evans rats, minor HapII-protected bands were also observed in ovary and uterus (arrows).


View larger version (52K):
[in this window]
[in a new window]
  		Fig. 6.   Mapping methylation status by sodium bisulfite modification method. A, numerous CpG loci (bars) constituted a CpG island in the rat cyclin D1 5'-flanking region. Sense and antisense primers (arrows) were set to cover the CpG loci located in the basic promoter region. B, high sensitivity mapping of the methylated cytosine was carried out by sodium bisulfite modification before PCR amplification to access more precise information about the methylation status, including non-CpG methylcytosine in various cell lines and tissues. In the K4D cell line, methylated cytosines are seen in 20 CpG loci. In K4D16 and K4DT leukemic cell lines, a part of the methylated cytosines, including the 5th CpG located between the two continuous Sp1-binding sites, is demethylated. Among normal rat tissues, the occurrence of methylated cytosines was less frequent than that among rat leukemic cell lines, and partial methylation of the cytosine at non-CpG loci around two continuous Sp1-binding sites (asterisk in A and B) is seen in the ovary and uterus. C, mapping of the methylated cytosine around the two continuous Sp1-binding sites (underlined) is shown. A methylated cytosine (arrow), corresponding to the 5th CpG locus in panel B, was seen in the K4D cell line, and demethylated in the K4D16 cell line.


View larger version (114K):
[in this window]
[in a new window]
  		Fig. 7.   Cyclin D1 expression by immunostaining and methylation status from microdissected samples in the rat uterus. The rat uterus was fixed with 4% paraformaldehyde, and embedded in paraffin. The thin sections were subjected to immunohistochemical staining for cyclin D1 protein. A, hematoxylin and eosin staining of the thin section (× 40). B, immunohistochemical staining for cyclin D1 protein using a serial section, boxed area in A (× 100). Positive immunoreactions are observed in the nuclei of the endometrial stromal cells in the functioning layer (1 and 2) but not in stromal cells in the basal layer and myometrium (3 and 4). C, to correlate the expression of cyclin D1 protein and the status of methylated cytosine, tissue sections were microdissected from endometrial stromal cells in the functional layer (1 and 2) and the basal layer (3 and 4), and the methylated cytosine was analyzed by agarose bead-mediated high sensitivity mapping. The dissected component from stromal cells in the basal layer (3 and 4) showed methylated cytosines at non-CpG loci in the first Sp1-binding site. On the other hand, the dissected component from proliferating stromal cells in the functional layer (1 and 2) showed either hemi-methylated (2) or non-methylated cytosines (1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we analyzed the epigenetic mechanism regulating cyclin D1 expression in rat leukemic cell lines and normal tissues. Two substrains of leukemic cell lines, K4DT (29) and K4D16 with monocyte-macrophage characteristics, were established from K4D (28), a chemically induced Long-Evans rat pluripotent leukemic cell line; the two substrains constitutively overexpressed cyclin D1 at both the mRNA and protein levels. To analyze the mechanism of cyclin D1 gene overexpression in K4DT and K4D16 cells, the 5'-regulatory region of the cyclin D1 gene from Long-Evans rat genomic library was cloned and characterized; the methylation status of the cytosine, especially around the two continuous Sp1-binding sites, was found to affect the steady-state expression of cyclin D1 not only in the leukemic cell lines but also in normal endometrial stromal cells.

Recent studies have demonstrated that the structure of cyclin D1 promoters of different species invariably have a core TATA-less promoter, containing two continuous Sp1-binding sites and a CRE, with high basal transcriptional activity in many types of cells (4, 11, 12, 35-39). Many activating mechanisms of cyclin D1 transcription through its promoters have also been reported, and some growth factors regulate cyclin D1 gene expression through species-specific regulatory elements and others through the common core promoter that is invariably present in mouse, rat, and human: in chondrocytes, the cyclin D1 gene has been identified as a target of activating transcription factor 2, which together with CREB, acts through CRE in human cyclin D1 gene core promoters (36); in the PC12 pheochromocytoma cell line, the nerve growth factor exerts its cell proliferative effect mainly through the Sp1 sites in the rat cyclin D1 core promoter (8). On the other hand, transformed p21ras and c-Ets-2 activate the human cyclin D1 promoter through the AP-1-like sequence located -954 base pairs upstream of the transcription start site (37), suggesting that, besides gene translocation or rearrangement in parathyroid tumor and mantle zone B-cell lymphoma, activation of oncogenes themselves directly affect the cell cycle regulatory pathway in some neoplasias. Furthermore, by electrophoretic mobility shift assay, the c-Fos and c-Jun bound AP-1-like sequence at -954 has been described as an enhancer sequence through which angiotensin II (40) and simian virus 40 small tumor antigen (41) transactivate the human cyclin D1 gene. We were, however, unable to locate this AP-1-like structure at least within the 2-kilobase upstream sequences in the Long-Evans rat cyclin D1 gene, and as shown in Fig. 4, transient transfection studies using unstimulated leukemic cell lines did not reveal any apparent enhancer sequences. We also noticed that there were species and subspecies differences of the promoter structures even in an essential gene as cyclin D1. Location of the putative E2F-binding site in the rat cyclin D1 promoter is, for example, different from that of the human (12), and even among the same rattus species (8), the putative binding sequence for the E2F family is different: TTTCGTGG for the Long-Evans rat and TTTCGGGG for the Wister rat (8). Since E2F-1 inhibits and E2F-4 induces cyclin D1 gene expression, and Sp1, Sp2, Sp3, and Sp4 show different affinity and activation properties for cyclin D1 gene expression (38), strict species- and subspecies-matched transfection studies are essential to assess the cyclin D1 gene promoter property especially in its steady state. Our study revealed that the shortest construct containing a CRE and the first Sp1-binding site (-105) showed prominent promoter activity (Fig. 4), indicating that the core promoter is important and essential for the steady-state expression of the cyclin D1 gene.

Since Southern blot and sequencing analysis with genomic DNA extracted from K4D16 and K4DT cells showed neither cyclin D1 gene amplification nor mutation in the promoter region (data not shown), the difference of the transcriptional machinery was further analyzed by transient transfection studies. Despite the difference in the rate of cyclin D1 gene transcription assessed by the nuclear run-on assay (Fig. 1C), transient transfection studies using a series of deletion constructs showed no significant difference in the steady-state promoter activity among the K4D, K4D16, and K4DT cell lines in a steady state (Fig. 4). Moreover the CpG loci in the cyclin D1 promoter region showed heavy methylation in K4D, and the 5th CpG located between two continuous Sp1-binding sites (underlined in Fig. 6C) was involved in in vitro demethylation in K4D16 and K4DT. These observations led us to speculate that an epigenetic mechanism controlling the expression of the rat cyclin D1 gene was present. Two major mechanisms of transcriptional suppression by CpG methylation at Sp1-binding sites have been postulated: as a direct inhibitory effect, cytosine methylation at the CpNpG site inhibits Sp1 binding in the CpG island of the retinoblastoma gene (42); another indirect mechanism of repression of transcription by MeCP2 (17) has also been demonstrated in the human leukosialin gene promoter (43). In this study, since CpG methylation was not within but adjacent to the Sp1 site in the K4D cells, and since non-CpG and non-CpNpG cytosine methylation within two continuous Sp1-binding sites was observed in the endometrial stromal cells, Sp1 binding to the rat cyclin D1 promoter was most likely repressed by the indirect mechanism.

Methylation status is developmentally organized; in the apolipoprotein A-I gene, a CpG island within the gene becomes demethylated in the early embryo, remains unmethylated through the late blastocyte stage, becomes methylated at the non-CpG sites, and then gradually undergoes demethylation in a tissue-specific manner (44, 45), postulating two different demethylation mechanisms: genome-wide nonspecific and site-specific demethylation. Moreover, for the latter mechanism, active demethylation by demethylase-transcriptional factor complexes (46) and passive demethylation by the binding of transcriptional factors to replicating DNA (47) are thought to be involved. That endometrial stromal cells in the functional layer (Fig. 7B, 1 and 2) are derived from those in the basal layer (Fig. 7B, 3 and 4) by a series of cell divisions (48) suggests that replication is essential for efficient de novo demethylation in rat endometrial stromal cells. Furthermore, since hemi-methylated cytosines were observed only in the transitional zone (Fig. 7B, 2 and 3) and then found totally demethylated in the superficial functional layer, a passive demethylation mechanism by an Sp1 transcriptional factor for preventing methylase from acting on post-replicative hemi-methylated DNA at Sp1-binding sites in the rat cyclin D1 gene is suggested. Since in the passive demethylation process Sp1-binding sites remain methylated in part of the cell population (47), methylated cytosines at Sp1-binding sites in the rat cyclin D1 gene may be requisite for keeping a number of the stromal cells alive against estrogen-induced proliferation that results in either apoptosis or compaction, then in deciduation and detachment (47).

In conclusion, we characterized the rat cyclin D1 gene 5'-flanking region and found that a basic promoter structure, with a CRE and two continuous Sp1-binding sites, was crucial for the steady-state expression by species- and subspecies-matched transfection studies, and that the steady-state expression of the rat cyclin D1 gene was epigenetically regulated by the cytosine methylation status especially around two continuous Sp1 sites in leukemic cell lines. Physiologically, in normal rat endometrial stromal cells, methylated cytosines at non-CpG loci around two continuous Sp1-binding sites were observed in the basal layer; they were then demethylated in the functioning layer where cyclin D1 expression was prominent.

    ACKNOWLEDGEMENTS

We thank Shuichi Matsuda, Izumi Iwamoto, and Atsumi Kodan for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by Ministry of Education, Science and Culture of Japan Grants 09670226 (to S. K.) and 10670206 (to R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Second Department of Pathology, Kobe University School of Medicine 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: kitazawa@med.kobe-u.ac.jp; Tel.: 81-78-382-5481; Fax: 81-78-362-0297.

    ABBREVIATIONS

The abbreviations used are: CRE, cAMP response element; CREB, cAMP response element-binding protein; PCR, polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Sherr, C. J. (1993) Cell 73, 1059-1065[Medline] [Order article via Infotrieve]
2. Quelle, D. E., Ashumn, R. A., Shurtleff, S. A., Kato, J., Bar-Sagi, D., Roussel, M. F., and Sherr, C. J. (1993) Genes Dev. 7, 1559-1571[Abstract/Free Full Text]
3. Jiang, W., Kahan, N., Tomita, N., Zhang, Y., Lu, S., and Weinstein, I. B. (1992) Cancer Res. 52, 2980-2983[Abstract/Free Full Text]
4. Matusshime, H., Roussel, M. F., Ashmun, R. A., and Sherr, C. J. (1991) Cell 65, 701-713[CrossRef][Medline] [Order article via Infotrieve]
5. Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Cell 70, 993-1006[CrossRef][Medline] [Order article via Infotrieve]
6. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499-511[CrossRef][Medline] [Order article via Infotrieve]
7. Morgan, D. O. (1995) Nature 374, 131-134[CrossRef][Medline] [Order article via Infotrieve]
8. Yan, G-Z., and Ziff, E. B. (1997) J. Neurosci. 17, 6122-6132[Abstract/Free Full Text]
9. Prall, O. W. J., Saecevic, B., Musgrove, E. A., Watts, C. K. W., and Sutherland, R. L. (1997) J. Biol. Chem. 272, 10882-10894[Abstract/Free Full Text]
10. Perry, J. E., Grossmann, M. E., and Tindall, D. J. (1998) Prostate 35, 117-124[CrossRef][Medline] [Order article via Infotrieve]
11. Herber, B., Truss, M., Beato, M., and Muller, R. (1994) Oncogene 9, 1295-1304[Medline] [Order article via Infotrieve]
12. Motokura, T., and Arnold, A. (1993) Genes Chromosomes & Cancer 7, 89-65[Medline] [Order article via Infotrieve]
13. Yan, Y-X., Nakagawa, H., Lee, M-H., and Rustgi, A. K. (1997) J. Biol. Chem. 272, 33181-33190[Abstract/Free Full Text]
14. Brooks, A., Shiffman, D., Chan, C. S., Brooks, E. E., and Milner, P. G. (1996) J. Biol. Chem. 271, 9090-9099[Abstract/Free Full Text]
15. Henglein, B., Chenivesse, X., Wang, J., Eick, D., and Brechot, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5490-5494[Abstract/Free Full Text]
16. Hwang, A., Maiy, A., McKenna, W. G., and Muschel, R. J. (1995) J. Biol. Chem. 270, 28419-28424[Abstract/Free Full Text]
17. Lewis, J. D., Meehan, R. R., Henzel, W. J., Maurer-Fogy, I., Jeppesen, P., Klein, F., and Bird, A. (1992) Cell 69, 905-914[CrossRef][Medline] [Order article via Infotrieve]
18. Tate, P., Skarnes, W., and Bird, A. (1996) Nat. Genet. 12, 205-208[CrossRef][Medline] [Order article via Infotrieve]
19. Simmen, M. W., Leitgeb, S., Charlton, J., Jones, S. J., Harris, B. R., Clark, V. H., and Bird, A. (1999) Science 283, 1164-1167[Abstract/Free Full Text]
20. Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. (1999) Nat. Genet. 21, 103-107[CrossRef][Medline] [Order article via Infotrieve]
21. Sinclair, A. J., Palmero, I., Holder, A., Peters, G., and Farrell, P. J. (1995) J. Virol. 69, 1292-1295[Abstract]
22. Mao, L., Merlo, A., Bedi, G., Shapiro, G. I., Edwards, C. D., Rollins, B. J., and Sidransky, D. (1995) Cancer Res. 55, 2995-2997[Abstract/Free Full Text]
23. Herman, J. G., Jen, J., Merlo, A., and Baylin, S. B. (1996) Cancer Res. 56, 722-727[Abstract/Free Full Text]
24. Herman, J. G., Civin, C. I., Issa, J. P., Collector, M. I., Sharkis, S. J., and Baylin, S. B. (1997) Cancer Res. 57, 837-841[Abstract/Free Full Text]
25. Villuendas, 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., and Piris, M. A. (1998) Am. J. Pathol. 153, 887-897[Abstract/Free Full Text]
26. Chomet, P. S. (1991) Curr. Opin. Cell Biol. 3, 438-443[CrossRef][Medline] [Order article via Infotrieve]
27. Olek, A., Oswald, J., and Walter, J. (1996) Nucleic Acids Res. 24, 5064-5066[Abstract/Free Full Text]
28. Maeda, S., Uenaka, H., Ueda, N., Shiraishi, N., and Suglyama, T. (1980) J. Natl. Cancer Inst. 64, 539-546
29. Fujita, M., Takahashi, R., Kitada, K., Watanabe, R., Kitazawa, S., Ashoori, F., Liang, P., Saya, H., Serikawa, T., and Maeda, S. (1997) Cancer Lett. 112, 47-55[CrossRef][Medline] [Order article via Infotrieve]
30. Hamburger, A. W., and Salmon, S. E. (1977) Science 197, 461-463[Abstract/Free Full Text]
31. Kitazawa, S., Ross, F. P., McHugh, K., and Teitelbaum, S. L. (1994) J. Biol. Chem. 270, 4115-4200[Abstract/Free Full Text]
32. Tamura, K., Kanaoka, Y., Jinno, S., Nagata, A., Ogiso, Y., Shimizu, K., Hayakawa, T., Nojima, H., and Okayama, H. (1993) Oncogene 8, 2113-2118[Medline] [Order article via Infotrieve]
33. Kitazawa, S., Kitazawa, R., Tamada, H., and Maeda, S. (1998) Biochim. Biophys Acta 1443, 358-363[Medline] [Order article via Infotrieve]
34. Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1827-1831[Abstract/Free Full Text]
35. Muller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J., Eilers, M., and Strauss, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2945-2949[Abstract/Free Full Text]
36. Beier, F., Lee, R. J., Taylor, A. C., Pestell, R. G., and LuValle, P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1433-1438[Abstract/Free Full Text]
37. Albanese, C., Johnson, J., Watanabe, G., Eklund, N., Vu, D., Arnold, A., and Pestell, R. G. (1995) J. Biol. Chem. 270, 23589-23597[Abstract/Free Full Text]
38. Yan, Y-X., Nakagawa, H., Lee, M-H., and Rustigi, A. K. (1997) J. Biol. Chem. 272, 33181-33190
39. Watanabe, G., Lee, R. J., Albanese, C., Reutens, A., Vairo, G., Henglein, B., and Pestell, R. G. (1998) Mol. Cell. Biol. 18, 3212-3222[Abstract/Free Full Text]
40. Watanabe, G., Lee, R. J., Albanese, C., Rainey, W. E., Batlle, D., and Pestell, R. G. (1996) J. Biol. Chem. 271, 22570-22577[Abstract/Free Full Text]
41. Watanabe, G., How, A., Lee, R. J., Albanese, C., Shu, I-W., Karnezis, A. N., Zon, L., Kyriakis, J., Rundell, K., and Pestell, R. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12861-12866[Abstract/Free Full Text]
42. Clark, S., Harrison, J., and Molloy, P. (1997) Gene (Amst.) 195, 67-71[CrossRef][Medline] [Order article via Infotrieve]
43. Kudo, S. (1998) Mol. Cell. Biol. 18, 5492-5499[Abstract/Free Full Text]
44. Shemer, R., Walsh, A., Eisenberg, S., Breslow, J. L., and Razin, A. (1990) J. Biol. Chem. 265, 1010-1015[Abstract/Free Full Text]
45. Shemer, R., Kafri, T., O'Connell, A., Eisenberg, S., Breslow, J. L., and Razin, A (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11300-11304[Free Full Text]
46. Weiss, A., Keshet, I., Razin, A., and Cedar, H. (1996) Cell 86, 709-718[CrossRef][Medline] [Order article via Infotrieve]
47. Matsuo, K., Silke, J., Georgiev, O., Marti, P., Giovannini, N., and Rungger, D. (1998) EMBO J. 17, 1446-1453[CrossRef][Medline] [Order article via Infotrieve]
48. Moulton, B. C. (1994) Endocrinology 134, 1055-1060[Abstract]

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 JEMHome page
H. Liu, J. Huang, J. Wang, S. Jiang, A. S. Bailey, D. C. Goldman, M. Welcker, V. Bedell, M. L. Slovak, B. Clurman, et al.
Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma
J. Exp. Med., August 4, 2008; 205(8): 1843 - 1858.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
V. Kundumani-Sridharan, D. Wang, M. Karpurapu, Z. Liu, C. Zhang, N. Dronadula, and G. N. Rao
Suppression of Activation of Signal Transducer and Activator of Transcription-5B Signaling in the Vessel Wall Reduces Balloon Injury-Induced Neointima Formation
Am. J. Pathol., October 1, 2007; 171(4): 1381 - 1394.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
D. Wang, Z. Liu, Q. Li, M. Karpurapu, V. Kundumani-Sridharan, H. Cao, N. Dronadula, F. Rizvi, A. K. Bajpai, C. Zhang, et al.
An Essential Role for gp130 in Neointima Formation Following Arterial Injury
Circ. Res., March 30, 2007; 100(6): 807 - 816.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. Kitazawa and S. Kitazawa
Methylation Status of a Single CpG Locus 3 Bases Upstream of TATA-Box of Receptor Activator of Nuclear Factor-{kappa}B Ligand (RANKL) Gene Promoter Modulates Cell- and Tissue-Specific RANKL Expression and Osteoclastogenesis
Mol. Endocrinol., January 1, 2007; 21(1): 148 - 158.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Zhang, M. A. Warren, S. F. Shoemaker, and M. M. Ip
NF{kappa}B1/p50 Is Not Required for Tumor Necrosis Factor-Stimulated Growth of Primary Mammary Epithelial Cells: Implications for NF{kappa}B2/p52 and RelB
Endocrinology, January 1, 2007; 148(1): 268 - 278.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. Isomura, M. F. Stinski, A. Kudoh, T. Daikoku, N. Shirata, and T. Tsurumi
Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication
J. Virol., August 1, 2005; 79(15): 9597 - 9607.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. L. Hilton, Y. Li, E. L. Dunphy, and E. H. Wang
TAF1 Histone Acetyltransferase Activity in Sp1 Activation of the Cyclin D1 Promoter
Mol. Cell. Biol., May 15, 2005; 25(10): 4321 - 4332.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Liu, J. Wang, and E. M. Epner
Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences
Blood, October 15, 2004; 104(8): 2505 - 2513.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. T.-K. P