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[Cancer Research 65, 3624-3632, May 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Regulation of DNA Methyltransferase 1 by the pRb/E2F1 Pathway

Michael T. McCabe1,2, Joanne N. Davis1 and Mark L. Day1,2

1 Department of Urology and 2 Program in Cell and Molecular Biology, Michigan Urology Center, University of Michigan, Ann Arbor, Michigan

Requests for reprints: Mark L. Day, Department of Urology, University of Michigan, 6219 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0944. Phone: 734-763-9968; Fax: 734-647-9271; E-mail: mday{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor suppressor gene silencing by DNA hypermethylation contributes to tumorigenesis in many tumor types. This aberrant methylation may be due to increased expression and activity of DNA methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine to cytosines in CpG dinucleotides. Elevated expression of the maintenance DNA methyltransferase, DNA methyltransferase 1 (DNMT-1), has been shown in carcinomas of the colon, lung, liver, and prostate. Based on the nearly ubiquitous alterations of both DNA methylation and the retinoblastoma protein (pRb) pathway found in human cancer, we investigated a potential regulatory pathway linking the two alterations in murine and human prostate epithelial cells. Analysis of DNA methyltransferase levels in Rb–/– murine prostate epithelial cell lines revealed elevated Dnmt-1 levels. Genomic DNA sequence analysis identified conserved E2F consensus binding sites in proximity to the transcription initiation points of murine and human Dnmt-1. Furthermore, the Dnmt-1 promoter was shown to be regulated by the pRb/E2F pathway in murine and human cell lines of epithelial and fibroblast origin. In the absence of pRb, Dnmt-1 transcripts exhibited aberrant cell cycle regulation and Rb–/– cells showed aberrant methylation of the paternally expressed gene 3 (Peg3) tumor suppressor gene. These findings show a link between inactivation of the pRb pathway and induction of DNA hypermethylation of CpG island–containing genes in tumorigenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In addition to the extensively studied role of genetic alterations (i.e., deletions, amplifications, and translocations) in cancer initiation and progression, epigenetic modifications of DNA also play a critical role in these processes through the regulation of gene transcription. DNA methylation represents the only covalent modification of DNA observed in higher organisms and has been implicated in the regulation of gene expression during development, genomic imprinting, X chromosome inactivation, and silencing of parasitic transposable DNA elements (see ref. 1 for review). DNA methylation can occur on cytosines found in CpG dinucleotides and its presence is generally associated with transcriptional repression (2). This repression is likely due to the recruitment of chromatin-modifying enzymes [i.e., histone deacetylases (HDAC) and SWI/SNF family proteins] to methylated DNA by proteins containing methyl CpG binding domains, such as MeCP2 and the MBD family proteins.

DNA methylation in mammalian cells is regulated by a family of highly related DNA methyltransferase enzymes (Dnmt-1, Dnmt-3a, and Dnmt-3b), which mediate the transfer of methyl groups from S-adenosylmethionine to the 5' position of cytosine bases in the dinucleotide sequence CpG (3, 4). Dnmt-1 functions as the maintenance DNA methyltransferase in mammalian cells and is therefore responsible for accurately replicating genomic DNA methylation patterns during the S phase of the cell cycle (5). On the other hand, de novo methylation of DNA is believed to be performed by Dnmt-3a and Dnmt-3b, which possess both maintenance and de novo DNA methylation activity (4). However, both groups of enzymes have been shown to exhibit some levels of both maintenance and de novo methylation in vitro, suggesting that this classification of the DNMTs may be oversimplified (6).

All types of cancers have been shown to possess aberrant DNA methylation characterized by global genomic hypomethylation and, yet at the same time, localized areas of hypermethylation frequently within the promoter regions of tumor suppressor genes (e.g., E-cadherin, GSTP1, MLH1, and p16INK4a; refs. 7, 8). Confirming the importance of DNA methylation in tumorigenesis, studies in several tumor types, including those of bladder, kidney, and colon, have shown all three DNA methyltransferases to be overexpressed (9). Overexpression of Dnmt-1 in mouse embryonic stem cells led to genomic DNA hypermethylation and loss of imprinting at the Igf2 locus similar to that observed in cancer (10). Additionally, murine (NIH3T3) and human (IMR90/SV40) fibroblasts expressing exogenous Dnmt-1 exhibited many hallmarks of transformation, including spindle morphology, decreased contact inhibition, increased genomic methylation, increased growth in soft agar, and increased tumorigenicity (11, 12). These data suggest that increased DNA methyltransferase levels and activity affect the methylation status of genes critical to tumor formation.

The retinoblastoma protein (pRb) participates in a well-characterized cell cycle regulatory pathway that is controlled by cyclin-dependent kinases (cdk) and cdk inhibitors (13). In this pathway, pRb functions to restrict cell cycle progression late in G1 in response to growth inhibitory signals, such as growth factor depletion or contact inhibition. When the pRb pathway becomes inactivated through any one of several mechanisms (e.g., cyclin D1 amplification, cdk4 overexpression, or p16INK4a inactivation), homeostatic balance may be lost, thereby promoting aberrant proliferation of cells leading to tumor formation. This loss of homeostasis due to inactivation of the pRb pathway also results from aberrant management of other cellular functions regulated by pRb, including differentiation (14), survival/apoptosis (15), and senescence (16).

A role for pRb in tumor development has been convincingly established through several animal models. Although homozygous knockout of Rb is embryonic lethal in mice, Rb+/– mice are viable and exhibit a predisposition to develop pituitary and thyroid tumors (17, 18). Additionally, Rb–/– prostate tissues have been rescued from embryonic Rb–/– mice by tissue recombination (19). Consistent with the tumor predisposition of Rb+/– mutants, Rb–/– prostate grafts exhibited increased susceptibility to the development of atypical hyperplasia and cancer in the presence of testosterone and estrogen (19). Therefore, these studies support a critical role for pRb in the prevention of tumor development.

The majority of activities attributed to pRb are believed to result from direct interactions between pRb and either transcription factors or HDACs (20–23). However, pRb may also function through interactions with components of the DNA methylation machinery, as pRb and Dnmt-1 interacted directly in glutathione S-transferase pull-down and coimmunoprecipitation experiments (24, 25). Furthermore, pRb overexpression greatly diminished the ability of Dnmt-1 to bind DNA and thereby induced genomic hypomethylation (25).

Based on the well-characterized role of pRb as a transcriptional repressor of E2F target genes and the observation that DNA methyltransferase 1 (DNMT-1) is overexpressed in tumors that frequently possess disrupted pRb pathways, we hypothesized that the DNMT-1 gene might be under the control of pRb and E2F transcription factors. In this study, we used Rb+/+ and Rb–/– prostate epithelial cell lines rescued by tissue recombination (26) to study the effect of pRb pathway inactivation on DNA methyltransferase levels. The mouse and human Dnmt-1 promoters were found to contain E2F binding sites that were required for regulation by pRb and E2F1. In the absence of pRb, the Rb–/– cells exhibited increased Dnmt-1 levels, which correlated with the inactivation of a tumor suppressor gene by DNA hypermethylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and cell treatments. wtPrE and Rb–/–PrE cell lines were generated and propagated as described previously (26). Briefly, prostate tissues obtained through tissue recombination (19) were minced in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 0.01 mg/mL bovine pituitary extract (Sigma, St. Louis, MO), 0.5 µg/mL cholera toxin (Sigma), 2 µmol/L dexamethasone (Sigma), 5 ng/mL insulin-like growth factor-I (Collaborative Research, Bedford, MA), 10 ng/mL epidermal growth factor (Collaborative Research), penicillin/streptomycin (Life Technologies, Grand Island, NY), 2 mmol/L L-glutamine (Life Technologies), and 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenious acid in an ITS premix (Collaborative Research). Cultures were later maintained in RPMI 1640 supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT). LNCaP and PC3 (American Type Culture Collection, Manassas, VA) prostate cancer cell lines were maintained under identical conditions. NIH3T3 mouse fibroblast cells were maintained in DMEM containing 10% FBS.

Inhibition of DNA methyltransferase activity was accomplished by growing cells in 1.0 µmol/L 5-aza-2'-deoxycytidine (5-Aza; Sigma) diluted in culture medium for 5 days. Twenty-four hours after passaging cells, culture medium was replaced with medium containing 1.0 µmol/L 5-Aza and was replaced with fresh 5-Aza-containing medium 3 days later. Cells for serum-free treatments were grown in RPMI 1640 without FBS beginning 24 to 48 hours after passaging and continuing for 2 to 5 days.

Western blot analysis. wtPrE and Rb–/–PrE cells were harvested by mechanical disruption with cell scrapers followed by gentle centrifugation. Cell pellets were then lysed in appropriate volumes of lysis buffer [50 mmol/L Tris (pH 8.0), 120 mmol/L NaCl, 0.5% NP40, 1 mmol/L EGTA, 100 µg/mL phenylmethylsulfonyl fluoride, 50 µg/mL aprotinin, 50 µg/mL leupeptin, 1.0 mmol/L sodium orthovanadate] for 1 hour on ice. Cellular debris was then pelleted by centrifugation and supernatants were collected and quantitated using a Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were then separated on precast Tris-glycine SDS-polyacrylamide gels (Novex, Carlsbad, CA) and transferred to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were blocked for 1 hour at room temperature in TBST [10 mmol/L Tris (pH 8.0), 150-500 mmol/L NaCl, 0.1% Tween 20] containing 10% nonfat dry milk before being probed overnight at 4°C with primary antibodies diluted in 2.5% milk/TBST. Following washes in 2.5% milk/TBST, membranes were incubated for 1 hour at room temperature with appropriate horseradish peroxidase–conjugated secondary antibodies. Following a second set of 2.5% milk/TBST washes, membranes were developed with enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech). Primary antibodies were obtained as follows: actin (Santa Cruz, Santa Cruz, CA, sc-1615), cyclin E (Santa Cruz, sc-481), Dnmt-1 (Santa Cruz, sc-10221), p16INK4a (Santa Cruz, sc-1207), p19Arf (Abcam, Cambridge, MA, Ab80, Ab80), proliferating cell nuclear antigen (PCNA; Santa Cruz, sc-9857), and pRb (PharMingen, San Diego, CA, 554136).

RNA extraction and reverse transcription-PCR. Total cellular RNA was extracted from ~5 x 106 wtPrE or Rb–/–PrE with RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's directions. The resulting total RNA (2 µg) was reverse transcribed using the ThermoScript reverse transcription-PCR (RT-PCR) system (Invitrogen, Carlsbad, CA). Briefly, reverse transcription was done with ThermoScript reverse transcriptase at 55°C for 1 hour using an oligo(dT)20 primer (50 µmol/L). PCR was carried out using Taq DNA polymerase (Invitrogen), 0.25 µmol/L gene-specific primers, and 2 µL cDNA. Variable numbers of PCR cycles were done by denaturation at 94°C (30 seconds), annealing at 59°C (45 seconds), and elongation at 72°C (1 minute). PCR products were resolved on 1.2% agarose gels containing 0.5 ng/mL ethidium bromide (Fisher Biotech, Pittsburgh, PA) in 0.5x Tris-borate EDTA buffer (BioWhittaker). The gene-specific primers used are available on request.

Cloning of the Dnmt-1 promoter region. The Dnmt-1 promoter region was amplified from high molecular weight DNA, isolated from wtPrE or LNCaP cells, using primers designed from publicly available sequences (National Center for Biotechnology Information Entrez accession no. AB056445): mDnmt-1 promoter forward 5'-CTCGAGGAAGAGTCCAGATGGTGTCCT-3' and reverse 5'-AAGCTTGCAGGTTGCAGACGACAGA-3' and hDNMT-1 forward 5'-CTCGAGCTTCTCGCTGCTTTATCCCC-3' and reverse 5'-AAGCTTCTCGGAGGCTTCAGCAGAC-3'. Italicized sequences represent nucleotides added to the complementary sequences to generate unique restriction digest sites. The resulting PCR products (1,220 bp for mDnmt-1 and 340 bp for hDNMT-1) were gel purified, cloned into the pCR2.1 TA cloning vector (Invitrogen), excised from pCR2.1, and cloned in the pGL3-Basic luciferase reporter construct (Promega, Madison, WI) to generate -1220mDnmt1-pGL3 and -340hDNMT1-pGL3. A unique NheI restriction site within the mDnmt1 promoter region was used to generate -734mDnmt1-pGL3 from -1220mDnmt1-pGL3. -236mDnmt1-pGL3 was generated by PCR with the reverse primer described above for -1220mDnmt1 and the following forward primer: 5'-CTCGAGGGAGGTGGGTGGCGCCAG-3'.

Site-directed mutagenesis was used to mutate the putative E2F binding sites within the context of the –734mDnmt1-pGL3 construct. QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used according to the manufacturer's protocol. Briefly, primers containing the desired mutations were generated as follows: E2FA mutant 5'-CCAATTGGTTTCCGCGCATTGAAAAAAGCCGGGGTCTCG-3', E2FB mutant 5'-CCCCCTCCCAATTGGTTTTCAATGCGCGAAAAAGCCGG-3', and E2FAB mutant 5'-CCCCTCCCAATTGGTTTTCAATATTGAAAAAAGCCGGGGTCTCG-3'. Italicized bases are mutated from the wild-type sequence. All plasmids were confirmed to contain the desired mutations by DNA sequencing at the University of Michigan DNA Sequencing Core (Ann Arbor, MI).

Luciferase reporter assays. Cells were plated into six-well tissue culture plates at a density of 1 x 105 cells per well. Twenty-four hours later, cells were transiently transfected in triplicate with Tfx-50 (Promega) or FuGene6 (Invitrogen) according to the manufacturer's directions, with the designated combination of expression plasmids at 1.0 µg (unless otherwise noted), 1.0 µg luciferase reporter plasmid, and 0.5 µg pSV-ß-galactosidase (pSV-ß-Gal; Promega) for transfection normalization. Cells were harvested 48 hours after transfection, lysed, and analyzed for luciferase and ß-galactosidase activity with luciferase substrate and Galacto-Light Plus ß-galactosidase chemiluminescent reporter assay (Tropix, Foster City, CA). All samples were measured on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Relative luciferase activity was determined by calculating the ratio between luciferase activity and ß-galactosidase activity.

Northern blot analysis. Total RNA was isolated as described above and RNA (10-20 µg) was electrophoresed on 0.85% agarose gels containing 4% formaldehyde and 1x MOPS. RNA was then transferred to Duralon-UV nitrocellulose membranes (Stratagene) overnight via capillary action with 20x SSC at room temperature. Following transfer, RNA was UV cross-linked to the membranes that were then incubated for 30 minutes in prehybridization solution [7% SDS, 0.25 mol/L Na2HPO4 (pH 7.2)]. Radiolabeled probes were generated from RT-PCR products for the specified transcripts of interest. Primer sequences used to generate probe templates are available on request. Probe template was then radiolabeled with [{alpha}-32P]dATP with Prime-It II Random Primer Labeling kit (Stratagene). Membranes were then incubated overnight at 65°C in hybridization buffer containing 2.0 x 106 cpm/mL. The following morning, membranes were washed twice in 20 mmol/L Na2HPO4 (pH 7.2), 5% SDS followed by two washes in 20 mmol/L Na2HPO4 (pH 7.2), 1% SDS at 65°C. Membranes were then exposed to BioMax MS-1 autoradiography film (Kodak, Rochester, NY).

Bisulfite DNA sequencing. Total genomic DNA was isolated from wtPrE and Rb–/–PrE using the DNeasy Tissue kit (Qiagen) according to the manufacturer's directions. Genomic DNA (2 µg) was then modified with bisulfite with the EZ DNA Methylation kit (Zymo Research, Orange, CA) according to the manufacturer's directions. Bisulfite-modified DNA (2 µL) was then used as template DNA in a PCR reaction with primers specific for the bisulfite-modified paternally expressed gene 3 (Peg3) CpG island as follows: forward 5'-TTTTGTAGAGGATTTTGATAAGGAGG-3' and reverse 5'-AAATACCTCTTTAAATCCCTATCACC-3'. The resulting PCR product was then electrophoresed on a 1.2% agarose gel and the Peg3 band was excised, purified, and cloned into pCR2.1. Ligation product was transformed into XL-1 Blue Escherichia coli and at least 10 individual colonies were selected for analysis from Luria-Bertani ampicillin agar plates. Individual clones were then analyzed by DNA sequencing through the University of Michigan DNA Sequencing Core facility.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dnmt-1 is overexpressed in Rb–/– prostate epithelial cells. Two nearly ubiquitous features of cancer cells are aberrant DNA methylation patterns and inactivation of the p16/pRb/E2F pathway. To investigate a potential causal link between pRb inactivation and altered DNA methylation and DNA methyltransferase regulation, we analyzed the expression of the three DNA methyltransferases with known methyltransferase activity in wild-type (Rb+/+) and Rb–/– cell lines. Wild-type (wtPrE) and Rb–/– prostate epithelial (Rb–/–PrE) cell lines were generated from prostatic tissues rescued from embryonic control (Rb+/+) and Rb–/– mutant mice by tissue recombination (26). The genotypes of these cell lines have been confirmed previously by genomic PCR and Western blot (26). Semiquantitative RT-PCR analysis of Dnmt-1, Dnmt-3a, and Dnmt-3b transcripts revealed that whereas amplification of the Dnmt-3a and Dnmt-3b transcripts was identical between Rb+/+ and Rb–/– genotypes under log-phase growth conditions the Dnmt-1 transcript exhibited elevated levels in the Rb–/–PrE cell line (Fig. 1A). Furthermore, Northern blot analysis of equivalent quantities of total cellular RNA from wtPrE and Rb–/–PrE cells with a 32P-labeled Dnmt-1 cDNA probe confirmed a consistent 3- to 4-fold up-regulation of Dnmt-1 transcript levels in the Rb–/–PrE cells (Fig. 1B). Finally, considering that Dnmt-1 protein levels dictate DNA methyltransferase activity, Western blot analysis was used to determine if the protein was also up-regulated in Rb–/–PrE. Antibodies raised against Dnmt-1 also showed increased expression of the 195-kDa Dnmt-1 protein in the Rb–/– cells compared with the Rb+/+ cells (Fig. 1C). Therefore, in the absence of the pRb, Dnmt-1 mRNA and protein expression was elevated 2-fold or greater due to altered regulation at the transcriptional level.



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  		Figure 1. Dnmt-1 is overexpressed in Rb–/– prostate epithelial cells. A, RNA was isolated from wtPrE and Rb–/–PrE cells and used for semiquantitative RT-PCR analysis of endogenous Dnmt-1, Dnmt-3a, and Dnmt-3b mRNA levels. RT-PCR samples were analyzed after 25, 28, 31, and 34 cycles of amplification to ensure that samples were within the linear range of amplification when examined. B, total RNA isolated from wtPrE and Rb–/–PrE cells was analyzed by Northern blot with a 32P-radiolabeled probe from murine Dnmt-1 cDNA. 28S, ethidium bromide staining of 28S rRNA before transfer. C, whole cell extracts were prepared from wtPrE and Rb–/–PrE and analyzed by SDS-PAGE and Western blotting with antibodies specific to Dnmt-1 or actin.

 
Characterization of the Dnmt-1 promoter region. To study the transcriptional regulation of Dnmt-1, 734 bp upstream of the murine Dnmt-1 gene was cloned into the pGL3-luciferase reporter construct for analysis of promoter activity. Due to some debate regarding the transcriptional start site for Dnmt-1, we have denoted the ATG translation start site as +1 to avoid any confusion. This region of the promoter exhibited considerable promoter activity comparable with the potent cytomegalovirus promoter, which served as a positive control (Fig. 2A). The DNA sequence responsible for this promoter activity was further narrowed down by analyzing a 236-bp fragment of the Dnmt-1 gene encompassing ~100 bp of DNA sequence both upstream and downstream of the transcription initiation site. This minimal promoter retained 60% of the activity of the 734-bp promoter fragment (Fig. 2A), suggesting that significant promoter activity is contributed by this minimal Dnmt-1 promoter. Additionally, these Dnmt-1 reporter constructs showed a 2-fold increase in basal promoter activity in Rb–/–PrE cells compared with wild-type cells (data not shown), suggesting that these constructs accurately represent the activity of the endogenous Dnmt-1 promoter.



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  		Figure 2. Identification and characterization of putative E2F binding sites within the murine Dnmt-1 promoter. A, murine Dnmt-1 promoter sequences (734 or 236 bp upstream of the ATG) cloned into the pGL3-luciferase reporter construct were transiently cotransfected into wtPrE cells along with pSV-ß-Gal. Activities of a promoter-less pGL3 (-) and cytomegalovirus (CMV) promoter-driven pGL3 (+) are also depicted. Columns, mean; bars, 1 SD. B, transcription factor binding site analysis with MatInspector identified three putative E2F binding sites (underlined) in close proximity to the murine Dnmt-1 ATG (box). Alignment of the corresponding human DNMT-1 promoter sequences is also demonstrated. C, site-directed mutagenesis was used to mutate the conserved putative E2F binding sites A, B, or A and B together within the -734mDnmt1-pGL3 construct. Wild-type and mutant plasmids were transiently transfected into murine wtPrE cells with pSV-ß-Gal. Columns, mean; bars, 1 SD.

 
The pRb negatively regulates a variety of transcription factors through the recruitment of transcriptional repressor molecules (e.g., HDACs). To determine if loss of any of the known pRb transcription factor interactions might be responsible for the elevated levels of Dnmt-1 in Rb–/–PrE, the genomic DNA sequence flanking the transcription and translation initiation sites for the mouse and human Dnmt-1 gene was queried for putative transcription factor binding sites with MatInspector version 2.2 (27). This DNA sequence analysis identified two conserved sequences within the murine and human Dnmt-1 promoters, which exhibited considerable similarity to the consensus E2F binding site [TTT(C/G)GCGC; Fig. 2B]. The two putative murine Dnmt-1 E2F binding sites were compressed into a single overlapping site in human DNMT-1 similar to E2F binding sites identified in several other cell cycle–regulated genes (e.g., N-myc, E2F-1, DHFR, PCNA, UDG, and E1a; ref. 28). Putative E2F binding sites were also identified in 5' untranslated regions of the Dnmt-1 gene from chicken and Xenopus (data not shown).

To determine if these putative E2F binding sites contributed to the regulation of Dnmt-1 promoter activity, we used site-directed mutagenesis to mutate the two conserved E2F sites in the mouse promoter. In contrast to mutation of the E2FA site, which had minimal effect on basal Dnmt-1 promoter activity, mutation of the E2FB site led to a 45% reduction in activity (Fig. 2C). When both E2FA and E2FB sites were mutated simultaneously, a 65% loss of activity was observed, suggesting that the E2FB site, perhaps along with the E2FA site, encodes sequences normally bound by a positive transcriptional activator.

The Dnmt-1 promoter is regulated by the pRb/E2F pathway. Considering the role of pRb in repression of E2F transactivation and the putative E2F binding sites within the Dnmt-1 promoter of several species, we investigated the possibility that aberrant E2F transcriptional activation was affecting Dnmt-1 levels. Cells lacking pRb have been shown previously to exhibit aberrant E2F activity, as both Rb–/– fibroblasts and epithelial cells exhibit elevated levels of a subset of E2F target genes, including cyclin E, PCNA, and p107 (26, 29). To explore the role of E2Fs in the regulation of Dnmt-1 promoter activity, exogenous E2F1 was coexpressed in wild-type prostate epithelial cells with the -734mDnmt1-pGL3 reporter construct. E2F1 was used, as it is one of the three activating E2Fs (E2F1-3) and is the most well characterized of the E2F family members. Increasing levels of exogenous E2F1 led to increased levels of promoter activity, suggesting that E2F1 is capable of activating the Dnmt-1 promoter (Fig. 3A). The ability of E2F1 to activate the Dnmt-1 promoter was greatly reduced in the presence of a mutated E2FA, E2FB, or E2FAB site (Fig. 3A). These data suggest that E2F1 activation of the murine Dnmt-1 gene is partly dependent on the integrity of the two putative E2F binding sequences. Additionally, similar to the promoter of the well-characterized E2F target Dhfr, the Dnmt-1 promoter failed to respond to E2F1 DNA binding (E2F1E132) and transactivation (E2F11-284) mutants (30).



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  		Figure 3. Murine and human Dnmt-1 promoters are regulated by the pRb/E2F pathway. A, wtPrE were cotransfected with wild-type, E2FmutA, E2FmutB, or E2FmutAB versions of the -734mDnmt1-pGL3 construct in the presence of 0, 0.1, or 1.0 µg exogenous E2F1. B, schematic of the -340hDNMT1-pGL3 and -340D61hDNMT1-pGL3 reporter constructs used in (C) and (D). Circles, putative E2F binding sites; arrows, transcription initiation point. Base pair numbering is from the translation start site. C, -340hDNMT1-pGL3 (black columns) or -340{Delta}61hDNMT1-pGL3 (white columns) were transiently transfected into human 293T cells in the presence of 1.0 µg pcDNA3, 1.0 µg E2F1, or 1.0 µg PSM-Rb. D, wtPrE, NIH3T3, and human LNCaP cells were transiently cotransfected with mouse or human DNMT1-pGL3 reporter constructs and either 1.0 µg pcDNA3, 1.0 µg DN-E2F1, or 1.0 µg E1a. DN-E2F1 is a fusion protein of the E2F1 DNA binding and dimerization domains to the pRb A/B pocket domain. Columns, mean; bars, 1 SD.

 
As mentioned previously, the human DNMT-1 gene exhibited putative E2F binding sites that aligned with those found in the murine Dnmt-1 gene. To determine if the human DNMT-1 promoter was also E2F regulated, a region of 340 bp upstream of the ATG translation start site was cloned into the pGL3-luciferase reporter construct (Fig. 3B) and transfected into LNCaP human prostate adenocarcinoma cells and 293T human embryonic kidney cells. In these cell lines, exogenous E2F1 reproducibly activated the human DNMT-1 promoter 6-fold, whereas a constitutively active unphosphorylatable large pocket of pRb (amino acids 379-928; PSM-Rb; ref. 31) repressed DNMT-1 promoter activity (Fig. 3C; data not shown). To examine if the conserved E2F binding site was required for pRb/E2F-mediated regulation, the 61 bp of 3' promoter sequence containing this site was deleted from the -340 bp DNMT-1 promoter to generate -340{Delta}61 DNMT-1 (Fig. 3B). When E2F1 was cotransfected with this -340{Delta}61 DNMT-1 promoter, a 67% decrease in activation was observed (Fig. 3C). Similarly, in the absence of the E2F binding site, the constitutively active pRb had no effect on DNMT-1 promoter activity. Additionally, the -340{Delta}61 DNMT-1 construct exhibited a 58% reduction in basal promoter activity compared with the -340 DNMT-1 construct (data not shown). These data suggest that the human DNMT-1 gene, like the murine Dnmt-1 homologue, is both positively and negatively regulated by the pRb/E2F pathway.

To further support a role for the pRb/E2F pathway in Dnmt-1 promoter regulation, additional regulators of this pathway were assessed for their ability to control Dnmt-1 promoter activity. First, a dominant-negative (DN) E2F construct was cotransfected along with the wild-type Dnmt-1 reporter constructs in wtPrE cells, NIH3T3 murine fibroblasts, and LNCaP human prostate adenocarcinoma cells (Fig. 3D). This previously described DN-E2F possesses the DNA binding domain of E2F1 fused to the pocket domain of pRb and is capable of repressing transcription of E2F target genes (32). DN-E2F was capable of repressing the mouse and human Dnmt-1 promoter reporter constructs (Fig. 3D) as well as the well-characterized E2F-responsive DHFR-Luc reporter construct (data not shown) in all cell lines analyzed. Additionally, the adenovirus E1a oncogene, which binds to and inactivates pocket proteins, was used to generate an increase in free E2F activity. Adenovirus E1a potently activated transcription of the mouse Dnmt-1, human DNMT-1, and DHFR promoters in all cell lines analyzed (Fig. 3D; data not shown). Additionally, Dnmt-1 promoter activation by E1a was dependent on its pocket protein binding activity, as the E1a CXdl (33) mutant, which does not interact with pocket proteins, was unable to activate the promoter >2- to 3-fold.3 Taken together, these data indicate that the E2F DNA binding domain localizes to both murine and human DNMT-1 promoter sequences where it activates transcription in a pocket protein-regulated manner.

Dnmt-1 is regulated throughout the cell cycle by the pRb/E2F pathway. The DNMT-1 transcript has been shown previously to be a growth-regulated transcript (9, 34, 35). Based on these observations and our data demonstrating regulation of Dnmt-1 promoter activity by pRb and E2F1, we predicted that normal cell cycle regulation of Dnmt-1 may be altered in cells lacking pRb. When wtPrE and Rb–/–PrE cell lines were deprived of serum for 48 hours, both cultures growth arrested similarly with increased cell populations found in the G1 phase of the cell cycle and decreased cell numbers in S phase as shown by flow cytometry (data not shown). Following 48 hours of serum deprivation, Rb+/+ prostate cells exhibited decreased expression of Dnmt-1 transcripts as is typical for other E2F targets (Fig. 4). However, the same period of serum deprivation did not lead to any detectable decrease in Dnmt-1 transcript in the Rb–/– cell line (Fig. 4). These data suggest that pRb is required for proper cell cycle regulation of Dnmt-1 transcription, which may be crucial for maintenance of normal methylation patterns.



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  		Figure 4. Cells lacking pRb fail to maintain proper cell cycle–specific regulation of Dnmt-1. Wild-type and Rb–/– prostate epithelial cells were signaled to growth arrest by serum withdrawal for 48 hours (time –48 to 0). Cells were then stimulated at time 0 to reenter the cell cycle following replacement of 5% FBS. Total RNA was prepared from cells at the indicated time points and RNA (10 µg) from each sample was analyzed by Northern blot with 32P-labeled probes specific for Dnmt-1.

 
Stable overexpression of E2F1 leads to elevated Dnmt-1 levels. Based on aforementioned data, it seemed that Dnmt-1 levels were elevated in Rb–/–PrE cells due to increased levels of free E2F1 and loss of pRb/E2F1 repressor complexes. To test this hypothesis, we established wtPrE cell lines that stably overexpressed E2F1 under control of the cytomegalovirus promoter. Two E2F1-overexpressing cell lines were generated through selection for G418 resistance. Northern blot analysis confirmed increased expression of the E2F1 transcript in transfected cells (Fig. 5A). Additionally, although we have not been able to identify an antibody capable of detecting the mouse form of E2F1 protein, we were able to show increased levels of two E2F targets (cyclin E and PCNA) at the protein level (Fig. 5B). Investigation of Dnmt-1 levels in these E2F1-overexpressing cells revealed overexpression at both mRNA transcript and protein levels (Fig. 5A and B) compared with cells transfected with the pcDNA3 control vector. This result strongly suggested that the Dnmt-1 overexpression observed in the Rb–/–PrE cell lines was due transcriptional activation resulting from elevated levels of active E2F1.



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  		Figure 5. Stable overexpression of E2F1 leads to accumulation of Dnmt-1 mRNA and protein. wtPrE cells were transfected and selected for G418 antibiotic resistance to stably express mouse E2F1 or pcDNA3 vector control. A, total RNA was isolated from log-phase wtPrE-pcDNA3 and wtPrE-mE2F1 cells and RNA (10 µg) from each sample was analyzed by Northern blot with 32P-labeled probes specific for Dnmt-1, E2F1, or actin. rRNA, ethidium bromide–stained rRNA before transfer. B, protein lysates generated from log-phase wtPrE-pcDNA3 and wtPrE-mE2F1 cells were analyzed by Western blot with antibodies raised against Dnmt-1, cyclin E, PCNA, or actin.

 
Transcriptional repression of selective genes in Rb–/–PrE cells due to promoter hypermethylation. Elevated levels of DNA methyltransferases have been proposed as an important mechanism involved in tumor initiation and progression (36, 37). The mechanism underlying the DNA methyltransferase–associated tumor susceptibility is likely due to transcriptional repression of tumor suppressor genes through promoter hypermethylation. To determine if the elevated Dnmt-1 levels in Rb–/–PrE cells had any effect on gene expression, we analyzed microarray data comparing wtPrE cells with Rb–/–PrE cells for genes that were strongly down-regulated in Rb–/– cells. One candidate gene was Peg3, which is an imprinted gene whose protein product is required for translocation of the proapoptotic Bax protein from the cytoplasm to the mitochondria during apoptotic stimuli (38). Peg3 was down-regulated an average of 22.2-fold across multiple arrays (data not shown). The strong repression of Peg3 was confirmed by semiquantitative RT-PCR in multiple independently derived Rb–/–PrE cell lines (Fig. 6A). Reexpression of the Peg3 transcript following treatment of Rb–/–PrE cells with 1.0 µmol/L 5-Aza, a DNA methyltransferase inhibitor, for 5 days suggested that the transcriptional repression of Peg3 was due to DNA methylation (Fig. 6B). However, to directly show methylation of the Peg3 gene, we used bisulfite modification and DNA sequencing to analyze the methylation status of a 444-bp CpG island encoded within the promoter, first exon and first intron of this gene (Fig. 6C). Analysis of 10 to 14 individual clones revealed significant hypermethylation across all 29 CpG dinucleotides in the Rb–/–PrE cells compared with Rb+/+ cells (Fig. 6C). These data confirm that the Dnmt-1 overexpression in cells lacking pRb can lead to observable alterations in gene expression.



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  		Figure 6. Dnmt-1 overexpression in Rb–/–PrE cells is correlated with DNA hypermethylation-induced transcriptional silencing of Peg3. A, semiquantitative RT-PCR was done on total RNA lysates from wtPrE and three Rb–/–PrE cell lines with primers specific for Peg3 and HPRT. B, wtPrE and Rb–/–PrE cells were treated with 1.0 µmol/L 5-Aza for 5 days. Total RNA was isolated and semiquantitative RT-PCR was done as above. C, bisulfite modification and DNA sequencing was done on genomic DNA isolated from wtPrE and Rb–/–PrE. Following amplification of the Peg3 CpG island by PCR, ≥10 individual alleles were cloned and sequenced. Y axis, percentage of total clones sequenced per cell line showing methylation at each CpG dinucleotide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inactivation of the pRb has been associated with increased tumor susceptibility in several model systems (17, 19). The precise mechanism underlying this sensitivity to tumorigenesis has not yet been fully elucidated. Traditional views of pRb function would suggest that inactivation of pRb leads to deregulation of E2F target genes, such as cyclin E, thymidine kinase, and DNA polymerase {alpha}, which govern control of the cell cycle. However, more recent studies of E2F target gene expression in models of Rb inactivation suggest that although a burst of E2F activity may follow acute loss of the Rb gene (39) compensatory mechanisms involving the remaining pocket protein family members, p107 and p130, are sufficient to keep the vast majority of E2F target genes in check when pRb is inactivated (29, 40–42).

In this study, we have identified putative E2F binding sites within the promoter region of the mouse and human Dnmt-1 genes, which are sufficient to allow for pRb- and E2F-mediated control of transcription. Rb gene inactivation is frequently observed in tumors of many tissue types and is typically thought to play a causative role in tumorigenesis. To analyze Dnmt-1 expression in a model of Rb gene inactivation, we analyzed the DNA methyltransferase levels in Rb+/+ and Rb–/– prostate epithelial cells. These cells were generated from wild-type and Rb–/– prostate tissues, which have been shown previously to be uniquely sensitive to hormone-induced tumorigenesis specifically in the absence of Rb (19, 26). Whereas no alterations were observed for Dnmt-3a or Dnmt-3b, we detected aberrant basal as well as cell cycle–specific regulation of Dnmt-1 in Rb–/– epithelial cells compared with wild-type controls. Additionally, this aberrant regulation of Dnmt-1 in Rb–/– prostate epithelium was correlated with increased DNA hypermethylation-mediated transcriptional silencing of Peg3, a gene involved in cell survival regulation.

Overexpression of DNA methyltransferases has been reported in many tumor types, including breast, colon, lung, and prostate cancers (36, 43–45). Suggestive of a functional role for DNA methyltransferase overexpression in cancer, increased expression of DNMTs in tumors has been significantly correlated with increased methylation of CpG islands found within the promoters of tumor suppressor genes (36, 37). Even more convincingly, several studies have shown that DNA methyltransferase levels and activity were progressively elevated in samples representing colon cancer progression with successively increasing activity levels from normal tissue from a normal patient to normal tissue from a cancer patient to polyps and finally to carcinoma (44, 46).

In addition to the descriptive and correlative studies on tumor samples, several genetic animal models have been used to implicate Dnmt-1 activity in tumor development. Although homozygous knockout of the Dnmt-1 gene results in embryonic lethality, several groups have generated hypomorphic Dnmt-1 alleles that typically express ~50% of normal DNA methyltransferase activity in heterozygotes (47, 48). Despite this decrease in DNA methyltransferase levels and activity, these animals remain viable and seem to maintain near normal levels of DNA methylation (48). Interestingly, when these animals with decreased Dnmt-1 levels were cross-bred with animal models of cancer, including the mismatch repair–deficient MLH1–/– and colon cancer–prone ApcMin/+ mice, significant decreases in tumor formation were observed compared with the single MLH1–/– or ApcMin/+ mutants (47, 49, 50). Additionally, Belinsky et al. showed a reduction by almost 50% in carcinogen-induced pulmonary hyperplastic and neoplastic lesions by inactivating one Dnmt-1 allele (51). Further repression of lesion formation by 89% was obtained when single allele Dnmt-1 inactivation was combined with 5-Aza treatment to inhibit remaining DNA methyltransferase activity (51). These animal model findings strongly suggest that DNA methyltransferase activity plays a causative role in the formation of these tumors.

In vitro studies have also shown a role for Dnmt-1 overexpression in the transformation process. As little as a 2-fold overexpression of exogenous DNMT-1 has been shown to lead to increased DNA methylation within the promoter regions of several tumor suppressor genes (i.e., HIC-1, ER, and E-cadherin) and tumorigenic transformation of cells as evidenced by altered cellular morphology, increased saturation density, and greatly increased growth in soft agar and tumorigenicity (11, 12, 52, 53). Similarly, treatment of transformed cells with the DNA methyltransferase inhibitor 5-Aza or with Dnmt-1 antisense oligonucleotides resulted in reexpression of previously methylated genes and reversion of the transformed phenotype (54). Interestingly, microarray analysis of a rat fibroblast cell line transformed by ~3-fold overexpression of the oocyte-specific Dnmt-1o (53) showed a similar pattern of transcriptional repression compared with our Rb–/– cell lines.4 For example, both cell lines exhibited down-regulation of Akap12 (Ssecks 322), glutathione S-transferase M2, growth arrest specific 5, embigin, protein S, membrane metalloendopeptidase, tissue plasminogen activator, etc. Considering the role of several of these genes in cell survival, signal transduction, and growth control, it is possible that through the combined effect of these transcriptional alterations, Rb–/– cells and Dnmt-1-overexpressing cells may be more susceptible to transformation and tumor induction.

In this study, we focused on the repression of Peg3 by hypermethylation. Peg3 is an imprinted gene with functions that are still not completely understood. However, inactivation of Peg3 by hypermethylation likely confers a survival advantage, as Peg3 regulates the translocation of the proapoptotic Bax from the cytoplasm to the mitochondria (38). Peg3 hypermethylation has been reported in gliomas and reexpression of a Peg3 cDNA in glioma cell lines resulted in a loss of tumorigenicity in nude mice, suggesting that this gene product functions as a tumor suppressor (55). Taken together, this epigenetic alteration would seem to provide Rb–/– cells with cell survival advantages compared with wild-type cells. Further investigation is necessary to determine if Peg3 transcriptional silencing is strictly required for tumor susceptibility of Rb–/– epithelium.

In support of an E2F-mediated mechanism of Dnmt-1 gene regulation, levels of Dnmt-1 increase during the transition from late G1 phase to early S phase in apparent preparation for the S-phase synthesis of DNA, which is initially unmethylated (56, 57). Additionally, Friend murine erythroleukemia cells were shown to express three species of DNA methyltransferases, one of which migrated at ~190 kDa similar to Dnmt-1 and increased during log-phase growth when many cells were involved in DNA synthesis (34). These observations suggested that E2F transcription factors might regulate Dnmt-1 levels considering that E2Fs are known to regulate many genes involved in DNA synthesis and S-phase progression (c-myc, E2F-1, p107, cyclin E, cyclin A, cdc2, Dhfr, PCNA, thymidine kinase, etc.). More recently, a study examining cell cycle–specific gene expression identified Dnmt-1 as part of a G1-S cycle cluster, which included several other E2F target genes, including cdk2, cyclin E, MCM3/5/6, PCNA, replication factor C, DNA polymerase I, and p107 (58). Thus, there is considerable evidence to support the cell cycle–specific regulation of Dnmt-1.

Microarray studies have recently become useful for identifying nearly comprehensive lists of target genes of various transcription factors. Recent reports using this technology to identify lists of pRb (59) or E2F (60) responsive genes have identified Dnmt-1 as being regulated by these proteins. Specifically, an inducible and constitutively active large pocket of pRb repressed Dnmt-1 ~2- to 3-fold in a Rat16 cell line, whereas E2F1 induced Dnmt-1 mRNA 2.2- to 2.4-fold in Rat-1a cells (59, 60). Dnmt-1 gene regulation seems to be not only under the control of pRb and E2F1 but also may be regulated by all of the activating E2Fs (E2F1-E2F3) and by the other pocket proteins (p107 and p130). Microarray studies examining gene expression alterations in response to p130 and E2F3 found Dnmt-1 mRNA to be down-regulated 3.5- to 4.9-fold following p130 expression and activated 3.2- to 4.3-fold by E2F3 (60, 61). Although these studies were not specifically designed to identify direct targets of pocket proteins and E2Fs, these data support our findings that Dnmt-1 is a direct transcriptional target of the pocket protein and E2F pathway.

We have shown that the Dnmt-1 gene is transcriptionally regulated by E2F transcription factors and that the normal expression of Dnmt-1 is kept in check by the pocket proteins. In the absence of pRb, cells exhibit increased transcription of the Dnmt-1 gene leading to accumulation of Dnmt-1 protein. These increased levels of Dnmt-1 were correlated with aberrant DNA hypermethylation-mediated transcriptional silencing of a tumor suppressor gene, which may predispose Rb–/– cells to tumorigenesis. Perhaps this ability of Dnmt-1 to be regulated by the pRb/E2F pathway explains the apparent lack of mutation or amplification of the Dnmt-1 gene in cancers, as pRb inactivation is nearly ubiquitous in cancer.


   Acknowledgments
 
Grant support: NIH grant R01 DK-61488 (M.L. Day), NIH Cancer Biology Training Fellowship grant 5T32 CA09676 (M.T. McCabe), and American Cancer Society-Clyde Dixon Trust (PF-03-252-01-TBE).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Peggy Farnham, Douglas Cress, Eric Knudsen, and Jean Wang for providing plasmids and reagents and Michael Imperiale and Colleen Doyle for critical review of this article.


   Footnotes
 
3 M.T. McCabe, J.A. Low, M.J. Imperiale, and M.L. Day, unpublished data. Back

4 M.J. McCabe and M.L. Day, unpublished data. Back

Received 6/18/04. Revised 12/20/04. Accepted 2/ 4/05.


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 Introduction
 Materials and Methods
 Results
 Discussion
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J. N. Davis, K. J. Wojno, S. Daignault, M. D. Hofer, R. Kuefer, M. A. Rubin, and M. L. Day
Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer
Cancer Res., December 15, 2006; 66(24): 11897 - 11906.
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 CarcinogenesisHome page
M. A. Knowles
Molecular subtypes of bladder cancer: Jekyll and Hyde or chalk and cheese?
Carcinogenesis, March 1, 2006; 27(3): 361 - 373.
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M. T. McCabe, J. A. Low, S. Daignault, M. J. Imperiale, K. J. Wojno, and M. L. Day
Inhibition of DNA Methyltransferase Activity Prevents Tumorigenesis in a Mouse Model of Prostate Cancer
Cancer Res., January 1, 2006; 66(1): 385 - 392.
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