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DNMT3B Variants Regulate DNA Methylation in a Promoter-Specific Manner

Departments of ¹ Thoracic/Head and Neck Medical Oncology and ² Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and ³ Department of Oncology, Beijing Cancer Hospital, Beijing University School of Oncology, Beijing, China

Requests for reprints: Li Mao, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Box 437, Unit 432, Room FC9.3065, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-792-1220; E-mail: lmao{at}mdanderson.org.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, DNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. DNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of DNMT3B4 in primary lung cancer, suggesting a role of DNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of DNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of DNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected DNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and DNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation. [Cancer Res 2007;67(22):10647–52]

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
DNA methylation plays an essential role in the normal development of the mammalian embryo by regulating gene transcription through genomic imprinting, X chromosome inactivation, and genomic stability (1). It is believed that DNA methylation patterns in somatic cells are established during gametogenesis and early embryonic development via consecutive waves of demethylation and de novo methylation (2). The DNA methyltransferase 3 (DNMT3) gene consists of DNMT3A and DNMT3B and is the major de novo DNA methyltransferase that preferentially methylates cytosine in CpG sites (3). Methylation in CpG-rich promoter regions may result in transcriptional silencing of the corresponding genes, which is a major mechanism by which tumor suppressor genes are inactivated in tumorigenesis (4).

DNMT3B contains 24 exons spanning 47 kb of genomic DNA. Two alternative 5' exons are used, but the same full-length DNMT3B protein (DNMT3B1 and DNMT3B2) is expected from both transcripts (5). Four additional transcriptional variants (DNMT3B3, DNMT3B4, DNMT3B5, and DNMT3B6) resulting from alternative pre-mRNA splicing have also been reported (5–7). Some of the variants may compete with each other, thereby resulting in even DNA hypomethylation (7). This possibility suggests a complex biological role of the DNMT3B variants. Increased expression of DNMT3B has been frequently observed in human cancer cell lines and primary tumors (3). However, an association between the expression level of DNMT3B and the promoter methylation status of tumor suppressor genes has not been established (8, 9). These data suggest that the regulation of DNA methylation of these promoters is complex.

DNMT3B, a subfamily of DNMT3B, consists of at least seven transcriptional variants by alternative pre-mRNA splicing (10). In non–small cell lung cancer (NSCLC), DNMT3B variants are the predominant forms of DNMT3B expression (10). We previously observed a strong and independent correlation between DNMT3B4 expression and DNA methylation of the RASSF1A promoter but not the p16 promoter (11). This finding suggested that DNMT3B variants are involved in the regulation of promoter methylation, possibly in a promoter-specific manner.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cell lines. Human NSCLC lines H1299 and H358 were purchased from the American Type Culture Collection. The HBE1 cell line was a gift from Dr. John Minna (The University of Texas Southwest Medical Center, Dallas, TX).

RNA extraction and reverse transcription-PCR. We isolated total RNA from cells by using Tri-Reagent (Molecular Research Center) according to the manufacturer's instruction. The primers used for reverse transcription-PCR (RT-PCR) were described previously (10).

Methylation-specific PCR. One microgram of genomic DNA was used for bisulfite treatment to modify unmethylated cytosine residues, and the modified DNA was used for methylation-specific PCR (MSP) using methylation-specific and unmethylation-specific primers as described previously (10, 11). Unmodified DNA was used to test all the primer sets and we failed to observe any amplified DNA fragment in our experimental conditions.

Small interfering RNA and antisense RNA transfection. Small interfering RNA (siRNA) specifically targeted to the junction of exons 5 and 7 of DNMT3B was designed and synthesized chemically (Ambion). Both annealed siRNA and corresponding oligonucleotides of single strands were used. The sequences were 5'-CACGCAACCAGAGAACAAGUU-3' (sense) and 5'-CUUGUUCUCUGGUUGCGUGUU-3' (antisense) for the target sequence 5'-AACACGCAACCAGAGAACAAG-3'. siRNA specifically targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or scramble siRNA was also obtained from Ambion to serve as controls.

Bisulfite sequencing of the RASSF1A promoter. MSP products were derived from H1299 cells treated with 40 nmol/L siRNA targeting DNMT3B4/2 or GAPDH for 24 h and were recovered by gel purification. The DNA fragments were cloned into a TA cloning vector (Invitrogen) according to the manufacturer's protocol. Plasmid DNA from each clone was then extracted, and inserts in individual clones were sequenced (T3 or T7 primer) using an ABI PRISM 377 DNA sequencer (Perkin-Elmer).

Western blot analysis. Cell lysates were obtained and equal amounts of protein from each sample were diluted with loading buffer, boiled, and loaded onto 7.5% SDS-polyacrylamide gel to be separated by electrophoresis followed by protein transfer to polyvinylidene fluoride membranes (Amersham). Proteins were detected by incubation with corresponding antibodies specific to either DNMT1 or V5 tag (Sigma) followed by blotting with horseradish peroxidase–conjugated secondary antibody (Sigma). The blots were then exposed to chemiluminescent substrate (Amersham) for detection.

Cell growth and cell cycle analyses. The ACEA RT-CES microelectronic cell sensor system (ACEA Biosciences) was used to confirm the number of living cells. The electronic sensors provided a continuous and quantitative measurement of the cell index (which depends on the number of attached cells and the shape of the cells) in each well. The cell cycle distribution of the cells was determined using a BD FACSCalibur flow cytometer and CellQuest software (Becton Dickinson).

Stable transfection. pcDNA6/V5-His (Invitrogen) was used to construct plasmids containing full-length DNMT3B2 or DNMT3B4. Empty vector or plasmids containing DNMT3B2 or DNMT3B4 were used to transfect HBE1 cells and establish clones with stable expression of the corresponding proteins. Several clones were selected from each transfectant, and passages 5 and 10 were subsequently used for promoter methylation analysis.

Bisulfite pyrosequencing. Pyrosequencing was used to quantitatively measure the levels of cytosine methylation of CpG sites of promoters as described previously (12). The primers used in this study are listed in Supplementary Table S1 and their locations in the CpG islands are presented in Supplementary Fig. S1. Assays were repeated thrice and the means of all experimental results were used with SEs. The quantification of cytosine methylation was performed using Pyro Q-CpG software (Biotage).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
To test the role of DNMT3B4 in the promoter-specific methylation of RASSF1A, we designed a siRNA that specifically targeted the junction of exons 5 and 7 of DNMT3B. Because both DNMT3B4 and DNMT3B2 lack exon 6, this siRNA is expected to trigger the degradation of both these transcripts. We used NSCLC cell line H1299 because it carries promoter methylation of both p16 and RASSF1A and expresses a high level of DNMT3B4 but no detectable DNMT3B (10).

We found that down-regulation of DNMT3B4/2 resulted in RASSF1A promoter demethylation in H1299 cells (Fig. 1 ). In the cells treated with 20 nmol/L or a higher concentration of the siRNA targeting DNMT3B4/2, a near complete RASSF1A promoter demethylation was observed as early as 12 h after treatment (Fig. 1A). This effect was maintained up to 72 h after treatment. The results are consistent with the dose-dependent reduction of DNMT3B4 expression by the siRNA or antisense treatment (Fig. 1B). In contrast, the promoter methylation status of p16 was not affected (Fig. 1A). These results provide the first direct evidence of a causal relationship between DNMT3B4 and the promoter methylation of RASSF1A in lung cancer cells.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Down-regulation of DNMT3B4/2 expression and demethylation of RASSF1A promoter. A, promoter methylation status of p16 and RASSF1A at different time points following treatment as measured by MSP. –, negative control; +, positive control. U, unmethylated DNA; M, methylated DNA. B, expression of DNMT3Bs in H1299 cells treated with siRNAs at various time points using RT-PCR. C, expression of RASSF1A mRNA (234 bp) of the corresponding samples measured by RT-PCR. D, methylation status in the RASS1A promoter according to bisulfate sequencing analysis. Arrowheads, cytosines not converted to thymidines in the CpG sites (bottom panel of the sequences) because of the resistance of the methylated cytosines to bisulfate treatment. Line in the figure below the sequences represents a clone. Open dot, a CpG site not methylated; solid dot, a methylated CpG site. The left set of lines represents clones from cells treated with control, and the right set of lines represents clones from cells treated with siRNA targeting DNMT3B4/2.

DNMT3B4/2

DNMT3B4/2

DNMT3B4/2

In a separate experiment, we used pyrosequencing method to analyze DNA from H1299 cells treated with either 20 nmol/L scramble siRNA control or 20 nmol/L siRNA targeting DNMT3B4/2 24 h after treatment. The primers used in this experiment were designed to avoid amplification bias (Supplementary Table S1). We observed that that promoter methylation of RASSF1A was decreased from 94% in the control-treated to 33% in the siRNA-treated DNA, whereas no difference was observed in the p16 promoter between control-treated and the siRNA-treated DNA (Supplementary Fig. S2). These results indicate that knockdown of DNMT3B4/2 can reverse the methylated CpG sites in the RASSF1A promoter region. Our finding is unlikely due to the inhibition of DNMT1 because the protein expression level was not reduced in the H1299 cells treated with the siRNA (data not shown). To determine whether the RASSF1A promoter demethylation due to knockdown of DNMT3B4/2 is limited to H1299 cells, we performed the same experiments with NSCLC cell line H358. Similar to our results with the H1299 cells, the RASSF1A promoter became unmethylated after the siRNA treatment but no effect was observed on the methylated p16 promoter (data not shown).

To address the issue whether some of the observed results are due to a shift in balance between DNMT3B4 and other DNMT3B isoforms, we also analyzed mRNA expression of DNMT3B5 and DNMT3B6 that are expressed in the H1299 cells beside DNMT3B1 that did not show change in expression levels after siRNA treatment (Fig. 1A). Interestingly, the expression of DNMT3B5 and DNMT3B6 was reduced in the siRNA-treated samples compared with the controls (Fig. 2A ). To ensure that the result was not due to nonspecific knockdown by the siRNA, we analyzed the expression of DNMT3B5 (DNMT3B6 was not expressed in the cell line) in HBE1 cells transfected with either DNMT3B2 or DNMT3B4. The expression of DNMT3B5 was increased in the cells transfected with either DNMT3B2 or DNMT3B4 compared with controls (Fig. 2B). The result indicates that the expression of either DNMT3B2 or DNMT3B4 may affect expression levels of DNMT3B5 and DNMT3B6.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Expression of DNMT3B5 and DNMT3B6 following change of DNMT3B4/2 expression levels. A, expression of DNMT3B5 and DNMT3B6 in H1299 cells treated with siRNAs measured by RT-PCR. B, expression of DNMT3B5 and DNMT3B6 in HBE1 cells transfected with DNMT3B2 or DNMT3B4.

DNMT3B4/2

DNMT3B4/2

DNMT3B4/2

DNMT3B4/2

DNMT3B4/2

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Biological effects of DNMT3B4/2 down-regulation. A, growth of H1299 cells transfected with siRNAs as measured using a microelectronic cell sensor system every 30 min. The representation of individual color lines is indicated and described in the figure. B, cell cycle distributions of H1299 cells 24 h after treatment with siRNAs as measured with a flow cytometer (a, cells treated with culture medium; b, Lipofectamine only; c, siRNA targeting GAPDH; d, scramble siRNA; e–g, 10, 20, and 40 siRNA, respectively; h, 40 nmol/L antisense RNA targeting DNMT3B4/2). Percentages of sub-G1 cells were labeled inside each panel.

DNMT3B4

DNMT3B2

DNMT3B2

DNMT3B4

DNMT3B2

DNMT3B4

DNMT3B2

DNMT3B4

DNMT3B4

DNMT3B2

DNMT3B4

DNMT3B2

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Overexpression of DNMT3B4 resulted in RASSF1A promoter hypermethylation. A, expression of recombinant DNMT3B4 and DNMT3B2 in HBE1 clones recognized on Western blots using antibody against V5 tag. HBE1-vec, vector only–transfected HBE1 cells. B, CpG methylation in promoter regions of six genes and in line sequences. Clones d3B4.C1, d3B4.C2, d3B4.C3, and d3B4.C4 expressed DNMT3B4, and clones d3B2.C1, d3B2.C2, and d3B2.C3 expressed DNMT3B2. The values for the genes represent the approximate percentages of the CpG sites that were methylated as measured by pyrosequencing analysis.

In a previous study, we found a statistically significant correlation between RASSF1A promoter methylation and DNMT3B4 expression in a large number of primary NSCLC tumors (11). That result provided in vivo evidence of a role for DNMT3B4 in regulating the methylation of CpG islands in a promoter-specific manner. The results presented in the current report provide enough direct evidence to establish the causal relationship between DNMT3B4 and RASSF1A promoter methylation but not between several other commonly methylated promoters we examined. In the siRNA-based experiment, the down-regulation of DNMT3B4 resulted in demethylation of the RASSF1A promoter but not the p16 promoter in two NSCLC cell lines. Because the siRNA used also knocked down DNMT3B2 (because of the shared exon-exon junction between DNMT3B2 and DNMT3B4), a role for DNMT3B2 in that process cannot be excluded. The experiments using HBE1 cells that express specific DNMT3B variants (DNMT3B2 or DNMT3B4) provided conclusive evidence that DNMT3B4 but not DNMT3B2 contributes to RASSF1A-specific promoter methylation. Although expression levels of DNMT3B4 may affect expression levels of DNMT3B5 and DNMT3B6, the expression of the later isoforms is unlikely contributed to RASSF1A promoter methylation because overexpressing DNMT3B2 also caused an increased expression of DNMT3B5 (Fig. 3B) but did not affect the methylation status of RASSF1A promoter (Fig. 4B).

Although our study results firmly establish the importance of DNMT3Bs in promoter-specific methylation, the detailed mechanisms are unknown. DNMT1 is the predominant cellular DNA methyltransferase, but it requires the participation of DNMT3B to achieve promoter methylation (17, 18). Because DNMT3Bs contain a PWWP domain, which has direct DNA-binding capability (19), the fact that there are DNMT3Bs with structural differences at and around the PWWP domain suggests that the DNMT3B variants interact with a class of promoters with a similar consensus sequence and are responsible for the methylation of the promoters. The recent finding that tumor-specific methylated genes have common sequence motifs in their promoters (20) supports this notion. It should be noted that, in our study, overexpression of DNMT3B4 in the HBE1 cells resulted in only partial methylation of the RASSF1A promoter; this observation indicates that an additional component or components are needed for the stable and complete methylation of the promoter. Alternatively, the peptide tags fused with DNMT3B4 may cause changes in protein folding and result in reduced efficiency of the protein.

Our findings place DNMT3Bs at the center of de novo promoter methylation, particularly in lung tumorigenesis. The promoter-specific demethylation we observed is particularly interesting for cancer therapy because it raises the possibility of inhibiting specific variants of DNMT3B to selectively activate critical tumor suppressor genes whose expression is down-regulated due to promoter methylation. Such an approach may lead to the development of novel therapeutic strategies tailored to individual tumors with particular epigenetic abnormalities. These strategies would cause limited adverse effects because normal tissue would be spared most of the effects of less targeted treatment on the promoters methylated.

	Acknowledgments

 
Grant support: Department of Defense grants DAMD17-01-1-01689-1 and W81XWH-05-2-0027.

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 Elizabeth L. Hess for scientific editing of the manuscript.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

J. Wang, M. Bhutani, and A.K. Pathak contributed equally to this work.

Received 4/11/07. Revised 8/30/07. Accepted 10/ 5/07.

	References

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