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Previous Article | Next Article 
Molecular and Cellular Biology, April 2006, p. 2976-2983, Vol. 26, No. 8
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.8.2976-2983.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Yasuhiro Yamada,1,,
Suzanne Nguyen,2,
Heinz Linhart,1
Laurie Jackson-Grusby,1,
Alexander Meissner,1
Konstantinos Meletis,1,¶
Grace Lo,1 and
Rudolf Jaenisch1,2*
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142,1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392
Received 26 October 2005/ Returned for modification 22 December 2005/ Accepted 11 January 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA methylation is catalyzed by a family of three DNA methyltransferases: Dnmt1, Dnmt3a, and Dnmt3b. Although the three Dnmts partially cooperate to establish and maintain genomic methylation patterns (21), they also have distinctive functions. Dnmt1 has a preference for hemimethylated DNA (1, 15, 38), and indeed a hypomorphic allele of Dnmt1 has been shown to cause global DNA hypomethylation (12). Dnmt1 is therefore considered the major maintenance methyltransferase. Dnmt3a and -3b probably function as de novo DNA methyltransferases because these enzymes were shown to have equal preferences in vitro for unmethylated and hemimethylated DNA (25, 26). Furthermore, de novo methylation of a subset of the CpG sites on stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein (17). Consistent with these notions, inactivation of both Dnmt3a and Dnmt3b by gene targeting blocks de novo DNA methylation in embryonic stem (ES) cells and early embryos, as well as de novo methylation of imprinted genes in germ cells (25, 26). These findings support the view that Dnmt3a and Dnmt3b function primarily as de novo methyltransferases during normal development. Nevertheless, the role of the de novo Dnmts in cancer is unresolved.
Evidence from humans and mouse models has shown that Dnmt3b is important for maintaining methylation of pericentromeric repetitive elements (5, 25) and of single copy genes in cooperation with Dnmt1 in a human colon cancer cell line (29, 37). Mouse embryonic fibroblasts expressing large T antigen and Ras form soft agar colonies and large tumors, but similarly treated fibroblasts from Dnmt3b–/– mice do not grow in soft agar and are much less tumorigenic in vivo (34). These findings suggest that deletion of Dnmt3b inhibits cell transformation. In addition, Dnmt3b deficiency promotes the chromosomal instability of mouse embryonic fibroblasts, which in turn promotes spontaneous immortalization or premature senescence (7). Furthermore, deletion of Dnmt3b was shown to induce apoptosis in a human cancer cell line (3). Taken together, these observations implicate a role for Dnmt3b in oncogenesis.
Despite these in vitro studies, there is no direct evidence in vivo to clarify the role of Dnmt3b in tumorigenesis. Here we show that Dnmt3b expression is elevated in colonic adenomas derived from the ApcMin/+ mouse model. We therefore chose this in vivo tumor model to analyze the role of Dnmt3b in tumor initiation and progression by using Cre-Lox mediated conditional Dnmt3b gene deletion in the intestinal mucosa. We found that the removal of Dnmt3b does not affect tumor initiation in the form of microadenomas but does inhibit the formation of macroscopic tumors at an early stage of tumor development.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A loxP site was inserted into the intron just 3' of the exon 19, which encodes DNA methyltransferase motif VI. In addition, a drug selection marker in the form of a neomycin phosphotransferase gene under the control of the phosphoglycerate kinase 1 gene promoter (pgk-neo cassette flanked by Frt sites) (28) was inserted into the intron between exons 15 and 16 of the Dnmt3b locus, which are 5' to the catalytic domain. The pgk-neo cassette was flanked by frt sites to facilitate removal by flp recombinase. A loxP site was also included just 5' to the pgk-neo cassette.
In the final targeting construct, pBS3blfN, the loxP sites flank exons 16 to 19, which encode the catalytic domains. Arms of genomic DNA from the Dnmt3b locus—a 1.9-kb HindII-EcoRV fragment on the 5' side and a 4.5-kb HindII fragment on the 3' side—surround the loxP-flanked region. The orientation of pieces in the targeting construct was confirmed by restriction digest analysis and sequencing.
Generation of ES cells and mice with conditional knockout alleles of Dnmt3b. Plasmid pBS3blfN was linearized with SacII and electroporated into F1 (129SvJae x C57BL/6; v6.5 line) mouse ES cells and selected for drug resistance against neomycin as described previously. Neomycin-resistant ES colonies were analyzed by Southern blotting. In ca. 45% of the picked colonies, the targeting construct had integrated properly into the Dnmt3b locus.
In order to remove the pgk-neo cassette, ES cells from a targeted clone were transiently transfected with enhanced flp (Flp-e) recombinase-encoding plasmids (a gift from Francis Stewart) (4) by lipofection with Cytofectene (Bio-Rad). Removal of the pgk-neo cassette was confirmed by PCR, Southern blotting, and by death in G418-containing medium (350 µg/ml). Sixteen of twenty-four subclones in this experiment no longer contained the neomycin resistance gene.
For injection into blastocysts, we used ES cells from one properly targeted subclone in which the neomycin resistance cassette was removed. The resulting chimeras were crossed with C57BL/6 mice to generate the founder colony.
Mice and polyp analysis. Mice were maintained in the facilities of Whitehead Institute for Biomedical Research. The conditional Dnmt3b allele was backcrossed to the C57BL/6 strain prior to crossing with ApcMin/+ mice. All tumor bearing mice analyzed in this experiment were backcrossed at least six generations into C57BL/6 mice. For tumor analysis, the entire intestine was excised immediately after sacrifice and subjected to a systematic microscopic screen for tumor formation along the entire length of the intestine. The investigator counting the adenomas was blind with regard to mouse genotype.
Genotyping. DNA was isolated from tail tips and amplified by PCR for the Dnmt3b locus as follows: Dnmt3bF (5'-GCTGAATTATACCCGCCCCAAGGAGGGCG-3'; located just 5' of the 3'loxP site) and Dnmt3bR (5'-CCCTTCAAAGGTGCATCGTGGCCAGTGTGC-3'; located in the 3' arm of the conditional construct). PCR products are 410 bp for the wild-type allele and 550 bp for the 2lox allele. For the Dnmt3b 1lox allele, Dnmt3bR was used in conjunction with Dnmt3b1F (5'-CCTGAGAGACTGGCCGGCTTTTCCTCAGAC-3'; located in the 5' arm of the conditional construct), generating a roughly 500-bp product from the Dnmt3b 1lox allele. C57BL/6 ApcMin/+ mice were purchased from Jackson Laboratories, Bar Harbor, Maine. Oligonucleotides RM21a (5'-TTCTGAGAAAGACAGAAGTTA-3'), RM20 (5'-TTCCACTTTGGCATAAGGC-3'), and RM19 (5'-GCCATCCCTTCACGTTAG-3' were used for genotyping. The Fabp-Cre mice (a gift from Jeffrey I. Gordon, Washington University, St. Louis, MO) were backcrossed to the C57BL/6 strain for more than 10 generations. Cre-positive mice were determined by PCR with the oligonucleotides CreF (5'-TGGGCGGCATGGTGCAAGTT-3') and CreR (5'-CGGTGCTAACCAGCGTTTTC-3').
Southern blotting, Western blotting, and histological analysis. Southern blotting, Western blotting, and histological analysis were performed as described previously (16). Total DNA was isolated from ES cells, intestinal mucosa, and tumor tissue and digested by BamHI for Southern blotting. The probe is a genomic fragment of Dnmt3b located downstream of the 3' end of the Dnmt3b targeting construct. Dnmt3b antibody (1:200 dilution for Western blotting; 1:100 for immunohistochemical analysis, IMGENEX), anti-ß-catenin antibody (1:1,000 dilution; BD Transduction Laboratories), and anti-Ki-67 antibody (1:250; Dako) were used in these analyses. The fluorescein in situ cell death detection kit, (Roche) was used to detect apoptosis.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To achieve intestine-specific deletion, a Cre transgene under the control of transcriptional regulatory elements from the fatty acid-binding protein gene (Fabp-Cre) (31) was used to generate Dnmt3b2lox/2lox, Fabp-Cre mice. Cre-mediated deletion of Dnmt3b was assessed by Southern blotting, which revealed ca. 50% recombination in the colon, but no recombination was detected in the small intestine from the same mice (Fig. 2C). To assess further the efficiency of Cre-mediated deletion within individual crypts, sections of the colon and small intestine of 8- and 21-week-old animals were stained with an anti-Dnmt3b antibody. Immunostaining showed ca. 50% of colon crypts stained positive for Dnmt3b in animals of both ages (Fig. 2D and 2E). Similar analysis of small intestinal crypts showed uniform expression of Dnmt3b in Dnmt3b2lox/2lox Fabp-Cre mice (data not shown), a finding consistent with the lack of recombination observed by Southern blotting in this tissue (Fig. 2C). Within the colon, immunostaining demonstrated that all cells of a given colon crypt were either Dnmt3b positive or negative, indicating that deletion of Dnmt3b likely occurred in crypt forming stem cells. Histological analysis did not reveal any significant phenotypic differences between Dnmt3b-positive and -negative crypts (Fig. 2D and E and data not shown).
Deletion of Dnmt3b decreases the number of colon tumors but not the formation of microadenomas. To study the effects of Dnmt3b deletion on intestinal tumorigenesis in ApcMin/+ mice, cohorts of 23 ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre+ and 18 ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre– mice were sacrificed at 150 days of age to quantify intestinal tumors. Since Fabp-Cre has different recombination efficiencies in different regions of the intestinal tract (Fig. 2C), tumor numbers in the small intestine and the colon were scored separately. Conditional deletion of Dnmt3b reduced the total number of colonic tumors by ca. 40% (P = 0.0012; Fig. 3A) as shown by the average colonic tumor burden of 2.75 ± 0.6 polyps in ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre+ mice, whereas ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre– mice had a mean macroscopic tumor load of 5.2 ± 0.7 colonic polyps. Based on the low recombination rate detected in the small intestine of Fabp-Cre mice, we anticipated little difference in tumor load in the small intestine of Fabp-Cre-positive and -negative cohorts. No significant difference was observed (P = 0.89, Fig. 3B), which shows the specificity of the result for Dnmt3b deletion in colon and supports the notion that Dnmt3b deficiency inhibits tumor formation in ApcMin/+ mice.
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Dnmt3b is not essential for tumor growth. The consistency in the frequency of Cre-mediated recombination within the colon (50%, Fig. 2C to E), along with the nearly twofold reduction in colonic polyps in the Cre-expressing cohort (Fig. 3A), suggested that deletion of Dnmt3b might provide a strong block to macroscopic tumor formation. To assess whether loss of Dnmt3b was compatible with macroscopic tumor growth, immunohistochemical staining of tumor sections using a Dnmt3b antibody was performed to determine the number of Dnmt3b-positive and -negative tumors. This analysis revealed that polyps from ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre+ mice were predominantly composed of both Dnmt3b-positive and -negative sectors, with a few sections showing homogeneous wild-type or mutant staining patterns (Fig. 4A and Table 1). To quantitate the extent of recombination throughout the tumors, we developed a competitive PCR assay to measure the ratio of 1lox to 2lox Dnmt3b alleles in DNA isolated from individual whole colon tumors (Fig. 4B). DNA samples from 2lox/2lox and 1lox/1lox ES cells were mixed in defined ratios to standardize the PCR conditions. This analysis revealed that most macroscopic tumors contained 2lox as well as 1lox Dnmt3b alleles, a few contained only the nonrecombined 2lox allele, and almost no tumors were observed with greater than 50% contribution of 1lox alleles.
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The presence of clonal sectors of Dnmt3b mutant cells within the macroscopic tumor lesions suggested that Dnmt3b function is dispensable in an already-formed colon tumor. To assess whether Dnmt3b deletion affects the proliferation rate of tumors, histological sections of colon polyps were stained with a cellular replication marker (Ki67 antibody). Consistent with the notion that Dnmt3b is not required in adenomas, no significant difference in Ki67 staining was observed between Dnmt3b-positive (45.7% ± 1.4% positive cells per crypt) and -negative (42.8% ± 1.45% positive cells per crypt) tumor sectors (Fig. 5A, P = 0.185). In addition, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed 0.90% ± 0.2% and 0.82% ± 0.3% TUNEL-positive cells/crypt in Dnmt3b-positive and -negative areas, respectively (P = 0.66), indicating there was no effect of the Dnmt3b deletion on tumor cell apoptosis (Fig. 5B). Furthermore, we calculated the tumor volume from ApcMin/+ Dnmt3b2lox/2lox mice that either carried or did not carry the Fabp-Cre transgene. Figure 5C shows that the average volume of colon tumors from ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre+ mice (11.84 ± 1.5 mm3) was not significantly different from the average tumor volume in ApcMin/+ Dnmt3b2lox/2lox Fabp-Cre– mice (11.04 ± 1.4 mm3; P = 0.68). We therefore conclude that Dnmt3b deficiency impairs the initiation of macroscopic tumors but has no noticeable effect on later stages of tumor growth (Fig. 6).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two findings indicate that Dnmt3b activity and, by extension, de novo methylation are required specifically at the transition from microadenoma to macroscopic tumor outgrowth. First, as summarized in Table 1, roughly 50% of microadenomas lacked Dnmt3b expression, which was equated to the recombination frequency in the colon. However, only 10% of macroscopic tumors developed without Dnmt3b, suggesting that the outgrowth of the Dnmt3b deficient microadenomas was selected against. Second, the subsequent maintenance of a tumor outgrowth was not dependent upon Dnmt3b since both DNA and immunohistochemical analyses indicated that most tumors in Fabp-Cre+ mice were mixed, being composed of cells carrying the unrecombined 2lox Dnmt3b allele as well as the deleted Dnmt3b 1lox allele. Together, these two results strongly suggest that Dnmt3b deletion selectively inhibits the transition from microadenoma to macroscopic adenoma and that the majority of macroscopic adenomas in these mice were initiated in cells with a nonrecombined Dnmt3b conditional allele (2lox). Persistent or stochastic expression of Cre recombinase within an already formed tumor may have allowed recombination to occur at some later point after adenoma outgrowth, which resulted in clonal expansion of Dnmt3b-negative sectors in a given adenoma. This interpretation is supported by immunostaining, which showed that the region of the tumor lacking Dnmt3b expression always appeared as coherent patches of variable size. Although immunohistochemical analyses indicated that ca. 10% of the adenomas lacked Dnmt3b expression, these were analyzed on representative sections and may even be an overestimate of the number of tumors that formed entirely lacking Dnmt3b (Fig. 4A). In a less biased analysis, DNA isolated from whole tumor samples failed to confirm the presence of a tumor with greater than 50% contribution by Dnmt3b mutant cells (Fig. 4B), and in fact most of the mixed tumors had a contribution of less than 20% based on the competitive PCR analysis. Together, these results suggest that Dnmt3b deficiency resulted in a more than three- to fourfold reduction in macroscopic tumor incidence, owing to a selective requirement for de novo methylation at the transition from micro- to macroadenoma (Fig. 6).
It has been suggested, based on the analysis of chimeric ApcMin/+ mice, that tumors in the small intestine arise by active interactions between crypts promoting loss of Apc and thus resulting in the formation of polyclonal adenomas (36). In that study, however, only 22% of all tumors were identified as polyclonal. A similar experiment analyzing chimeric ApcMin/+ mice, using lacZ as a reporter, yielded only 9% mixed tumors (22). Also, in our experimental system, an average of only three adenomas were observed in the colon, in contrast to an average of 17 tumors in the small intestine of the chimeric mice (36). Because the same frequency of recombination in crypts and microadenomas was observed in our study and the microadenomas were homogeneous in Dnmt3b expression (Table 1), our data indicate the initiating event of LOH at the APC locus must precede the local recruitment of a similarly mutated crypt to produce the mixed tumor class according to the collision model. The low incidence of lesions in our system virtually excludes the possibility that the random collision model accounts for a substantial fraction of the majority class of mixed tumors observed in the ApcMin/+ Dnmt3b conditional mutants. Consistent with this conclusion are other studies that also demonstrated a monoclonal origin of colon tumors in mice (27). We think it more likely that persistent and stochastic expression of Cre recombinase within colonic tumors, which we observed by reverse transcription-PCR, explains the formation of the mixed class of tumors in these animals.
It has been shown previously that global DNA hypomethylation can protect as well as enhance tumor formation (8, 12, 19). For example, reduced Dnmt1 activity was demonstrated to inhibit adenoma formation in ApcMin/+ mice but to promote formation of lymphomas in the thymus and of sarcomas in soft tissues (9, 12). Enhanced thymoma and sarcoma formation was due to increased genetic instability and elevated LOH frequencies of tumor suppressor genes. Consistent with this finding is our recent observation that reduced levels of Dnmt1 expression increased the number of microadenomas in ApcMin/+ mice (39) by enhancing the rate of Apc LOH (Fig. 6). In contrast to reduced Dnmt1 activity, the somatic deletion of Dnmt3b would be predicted to have no effect on global methylation levels and thus would not be expected to affect APC LOH. In agreement with this prediction is the finding that microadenoma formation is unaffected by loss of Dnmt3b (Fig. 6).
Protection against macroscopic tumors by reduced Dnmt1 expression was suggested to be due to an epigenetic mechanism such as the impaired maintenance of methylation marks that are acquired during tumorigenesis. For example, de novo methylation of tumor suppressor genes of the H19 DMR has been shown to promote intestinal tumor load by loss of imprinting of Igf2 (6, 33). Because the maintenance of methylation marks depends on Dnmt1 activity, a reduced Dnmt1 level may lead to loss of DMR methylation causing reduced Igf2 expression and thus protect against intestinal tumors. Our results indicate that somatic deletion of Dnmt3b also protects against polyp formation, suggesting that this de novo methyltransferase is causally involved in tumor formation, possibly by promoting de novo methylation that mediates silencing of tumor suppressor genes (35) or loss of imprinting of genes such as Igf2 (6, 33). This predicts that, once tumor growth is initiated, continuous expression of Dnmt3b would not be required for further tumor growth (Fig. 6). Indeed, we found that loss of Dnmt3b has no noticeable effect on the growth of established tumors as the proliferation and apoptosis rate of cells was similar in Dnmt3b-positive and -negative sectors of a given tumor. Also, we failed to detect any significant difference in the average tumor volume when comparing Dnmt3b2lox/2lox ApcMin/+ mice with or without the Fabp-Cre transgene, which is consistent with tumor growth being unaffected by Dnmt3b. These studies are consistent with the results obtained by targeted disruption of the human DNMT3B gene in the human colon cancer cell line HCT116, which resulted in little to no change in the global DNA methylation or growth of these cells. Although the disruption of both DNMT1 and DNMT3B caused DNA hypomethylation in the same colorectal cancer cell line, our results suggests that Dnmt1 alone is sufficient to support the proliferation and maintenance of tumors in ApcMin mice (29, 37). Thus, although Dnmt3b is required during the initial outgrowth phase of macroscopic colonic adenomas in these mice, it has no effect on subsequent tumor growth and maintenance (Fig. 6).
Our previous observation that inhibition of Dnmt1 protected against intestinal tumorigenesis (19) sparked efforts to develop inhibitors of Dnmt1 that could be administered prophylactically to FAP patients carrying an Apc mutation. However, the later recognition that Dnmt1 inhibition can impair genomic stability and enhance tumor formation in other tissues such as the thymus or in soft tissues (9, 12) cautioned against long-term treatment with Dnmt1 inhibitors. Since Dnmt3b inhibition has no effect on microadenoma formation and, by implication, on genomic stability but strongly inhibits macroscopic tumor initiation, drug-mediated inhibition of this enzyme may be effective as a prophylactic agent to protect against tumors without the potential side effects of Dnmt1 inhibitors.
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ACKNOWLEDGMENTS |
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H.L. was supported by a fellowship from the Canadian Institutes of Health Research. R.J. received support from the NIH (R37-CA84198, RO1-HD0445022, and RO1-CA87869).
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FOOTNOTES |
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H.L., Y.Y., and S.N. contributed equally to this study.
Present address: Department of Tumor Pathology, Gifu University, Japan.
Present address: Department of Pathology, Harvard Medical School, Boston, MA 02115.
¶ Present address: Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden.
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