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Previous Article | Next Article 
Molecular and Cellular Biology, June 2007, p. 4049-4057, Vol. 27, No. 11
0270-7306/07/$08.00+0 doi:10.1128/MCB.02023-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Alan Clarke,2 and
Adrian Bird1*
Wellcome Trust Centre for Cell Biology, Edinburgh University, The King's Buildings, Edinburgh EH9 3JR, United Kingdom,1 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom2
Received 29 October 2006/ Returned for modification 30 November 2006/ Accepted 6 March 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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80-amino-acid methyl-CpG binding domain (10), and the Kaiso-like proteins, Kaiso, ZBTB4, and ZBTB38, which bind DNA methylated at CpGs via a conserved zinc-finger motif (9, 27). Biochemical analysis of MeCP2, MBD1, MBD2, and Kaiso has provided evidence that each can associate with a specific corepressor complex and bring about transcriptional silencing in model gene expression systems.
The present study concerns MBD2, which is a transcriptional repressor that can be isolated in a complex with components of the NuRD complex, including the SNF2-like motor protein Mi-2 and histone deacetylases (8, 22, 26, 34). MBD2 is the only member of the MBD protein family to be represented by invertebrate orthologs (12). Evidence for the involvement of MBD2 in differentiation and development has come from mouse genetics. Mice in which the Mbd2 gene is deleted are viable and fertile (11), but they mis-express the interleukin 4 and interferon 
genes in T helper (TH) cells (13). Expression of the interferon and interleukin 4 genes is mutually exclusive in the TH1 and TH2 derivatives of wild-type naive TH cells, but Mbd2–/– TH cells often expressed both genes simultaneously. Failure to adequately repress these genes in differentiated TH1 and TH2 cells alters the immune response in MBD2-deficient animals (14). Also, aberrant derepression of human fetal globin genes in Mbd2-null mice bearing a transgene that contains the human ß-like globin gene cluster has also been reported (31). In addition to these examples of gene misregulation, it has been found that depletion of MBD2 greatly reduces the number of intestinal adenomas in tumor-prone ApcMin mice (32). Although the molecular basis of this effect is unknown, it may be relevant that MBD2 is required for silencing of specific tumor-suppressor genes in human colorectal carcinoma cells and in other human cancer cell lines (3, 24). MBD2-mediated repression of tumor suppressor genes may therefore be a prerequisite for tumor formation, although other explanations cannot currently be ruled out.
In the present study we investigated gene expression in the colons of Mbd2–/– mice. We found that a set of genes coding for digestive enzymes normally expressed only in duodenum and pancreas are highly overexpressed in the Mbd2-null colon, the level of overexpression being variable between individual mice. DNA methylation profiles were indistinguishable between wild-type duodenum and colon, but MBD2 was 5-fold more abundant in colon. The MBD2-deficient phenotype could be mimicked in human colorectal carcinoma cells by depletion of MBD2 or DNA methylation, leading to overexpression and altered chromatin structure at these genes. Our results establish that a stable gene expression pattern in the mature gut is dependent on DNA methylation and MBD2.
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MATERIALS AND METHODS |
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Cell lines. HCT116 cells were obtained from the American Type Culture Collection. DKO cells were a gift from B. Vogelstein. Both cell lines were maintained in RPMI medium containing 10% (vol/vol) fetal calf serum. For HDAC inhibition, cells were treated with 500 nM trichostatin A (Sigma) for 18 h.
MBD2 siRNA and shRNA. Two different predesigned small interfering RNAs (siRNAs) directed against MBD2 (ID no. 14015 and ID no. 13925) were purchased from Ambion. Both siRNAs (150 pmol each) were cotransfected three times at 24-h intervals into HCT116 cells using Oligofectamine (Invitrogen). Silencer negative control siRNA#1 (Ambion) was used as a control. To generate the small hairpin RNA (shRNA) vector, a 64-bp hairpin sequence directed against MBD2 was cloned into the pSuper vector with puromycin resistance. Stable cell clones were obtained after puromycin selection.
Real-time RT-PCR. Total RNA was extracted from mouse intestines using RNA-Bee (Tel-Test) and cultured cells using TriReagent (Sigma) according to the manufacturer's instructions. After DNase treatment with DNA-Free (Ambion), cDNA was transcribed using Moloney murine leukemia virus reverse transcriptase (Promega). Real-time PCR was carried out with iQ SYBR Green Supermix (Bio-Rad) on an iCycler (Bio-Rad) according to the manufacturer's instructions. The primer sequences used for reverse transcription-PCR (RT-PCR) can be obtained on request.
Western blotting and immunohistochemistry. Approximately 100 mg of snap-frozen mouse intestine was placed into 600 µl of Laemmli buffer (60 mM Tris-HCl, 100 mM dithiothreitol, 10% glycerol, 2% sodium dodecyl sulfate [SDS]), sonicated for 15 s, and boiled for 6 min. After spinning for 4 min at 14,000 rpm (4°C), the supernatant was run on a polyacrylamide gel. Western blot analyses with protein extracts from mouse tissue or cultured human cells were performed using standard protocols. Rabbit R593 anti-MBD2 antiserum (26) was used to probe the blots. Immunohistochemistry was performed on 5-µm cryosections fixed in 4% paraformaldehyde. After the sections were boiled in 10 mM citric acid (pH 6.0) for 5 min, staining was performed using the Mouse-on-Mouse peroxidase kit (Vector Laboratories) according to the manufacturer's instructions. A mouse monoclonal antibody (anti-human spasmolytic polypeptide, catalog no. NCL-HSP; Novocastra) was used to detect TFF2 (spasmolytic polypeptide).
Chromatin immunoprecipitation (ChIP). Cells were grown until confluent, cross-linked in 1% formaldehyde in phosphate-buffered saline (PBS) for 10 min, washed in PBS, and lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.0]). The chromatin was sonicated for 3 min at 35% amplitude using a digital sonifier (Branson) and then diluted 1:10 in immunoprecipitation dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.0]). After the samples were precleared for 2 h with salmon sperm DNA-bovine serum albumin-protein G-Sepharose beads, 15 µg of affinity-purified anti-MBD2 antiserum (S923 [26]), anti-acetyl histone H4 antibody (catalog no. 06 to 598, Upstate), or unrelated control antibodies were added to each sample, followed by rotation overnight at 4°C. To collect the immunocomplexes, 100 µl of protein G-Sepharose was added to the samples for 1 h at 4°C. The beads were then washed once in buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl), four times in buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl), once in buffer 3 (250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl), and twice in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Immunocomplexes were then eluted with 2 x 100 µl of extraction buffer (1% SDS, 100 mM NaHCO3), and cross-links were reversed by adding 5 M NaCl to a final concentration of 300 mM, followed by incubation at 65°C overnight. The primer sequences for amplification of the TFF2 promoter can be obtained on request.
Bisulfite genomic sequencing. DNA was phenol-chloroform extracted from cultured cells or mouse intestine after proteinase K digestion. DNA (2 µg) was bisulfite modified and PCR amplified (primer sequences are available on request). PCR products were gel purified and cloned using the TOPO TA cloning kit (Invitrogen). Colonies for each PCR (10-14) were picked for sequencing.
Global DNA methylation analysis. Decreasing amounts of DNA were slot blotted onto a nitrocellulose membrane and then incubated with recombinant protein consisting of four sequential hemagglutinin (HA)-tagged methyl-CpG-binding domains of MBD1, which can bind to methylated but not unmethylated DNA (16). Incubation with an anti-HA antibody allowed visualization and quantification of the amount of methylated DNA in the sample. M.Sss1-methylated DNA and DNA isolated from bacteriophage lambda were used as positive and negative controls, respectively, for CpG methylation.
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RESULTS |
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50%. Levels of overexpression also varied widely, with some animals expressing >10,000-fold more than the basal levels in wild-type colon. The results demonstrate a continuum of overexpression in the absence of MBD2.
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Differentiation of the exocrine pancreas is under the control of the transcription factor complex Ptf1, which comprises two widely expressed proteins, p64 and p75, plus the highly tissue specific factor p48 (19, 20, 30). Mice carrying a disrupted p48 gene specifically lack an exocrine pancreas (30). We sought to determine whether the overexpression of ExPa genes correlated with p48 mis-expression in MBD2-deficient colons. The results showed a strong correlation, since p48 expression was confined to colons that also overexpressed the pancreas-specific gene set (Fig. 1A). Derepression of p48 may therefore trigger the overexpression phenotype. We also tested the expression of Cdx1 and Cdx2, which are required for anterior-posterior patterning and the development of the intestinal epithelium. No significant difference in mRNA levels between wild-type and Mbd2-null colons was observed (data not shown).
To determine which regions of the Mbd2-null intestine overexpress ExPa genes, we isolated RNA from the colon and the three segments comprising the small intestine: the duodenum, the jejunum, and the ileum. Real-time RT-PCR revealed that ExPa genes are, as expected, expressed in the duodenum in wild-type mice (Fig. 1D). In Mbd2-null mice, aberrant expression of digestive enzymes was only observed in the colon, since the jejunum and ileum were not affected. At their highest, the expression levels of p48, Tff2, and Ela2 in the colons of Mbd2-deficient mice were comparable to levels seen in the duodenum. We next sought to determine whether variable ExPa gene expression in Mbd2-null mice was caused by different numbers of expressing cells per colon or different levels of expression in a constant set of cells. Strong cytoplasmic immunostaining for TFF2 was observed in goblet cells and enterocytes in two of five Mbd2-null mouse colons (Fig. 1E and data not shown). One ExPa-expressing colon showed TFF2 expression in multiple cells in adjacent crypts (Fig. 1E, middle panel), whereas in the other mouse strong staining was only seen in single cells in a small fraction of crypts (Fig. 1E, top panel). No TFF2-positive crypt cells were seen in five wild-type mouse colons (Fig. 1E, bottom panel, and data not shown). Variability of expression levels in Mbd2-deficient mice may therefore be due to different numbers of crypt cells that escape ExPa gene repression.
Since MBD2 is reported to read the DNA methylation signal, we speculated that reduced levels of DNA methylation would also induce inappropriate expression of the same genes. Absence of the maintenance DNA methyltransferase DNMT1 is lethal in mice (23), but phenotypic effects on cancer susceptibility have been observed in Dnmt1+/– heterozygotes, suggesting that reducing the amount of enzyme by half affects DNA methylation (21). To test for the effects of heterozygosity of the Dnmt1c allele on DNA methylation levels, we carried out bisulfite genomic sequencing of a region upstream of the Tff2 promoter. Average DNA methylation across the region was significantly less in Dnmt1c/+ heterozygotes than in wild-type mice (61% versus 38%; Fig. 2A). When the expression of ExPa genes was examined in Dnmt1c/+ mice, we observed overexpression of the ExPa genes in 4 of 15 mice (Fig. 2B). As in the case of Mbd2-null mice, Dnmt1 deficiency increased the probability that colon cells escape the repression of ExPa genes that normally prevails in this organ.
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5-fold higher in the colon than in the duodenum as determined by Western blot analysis when normalized against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein (Fig. 3D). Levels of MBD2 in the ileum and jejunum, where ExPa genes are also silent, were likewise higher than in the duodenum and were similar to colon levels (Fig. 3D). We only detected the larger MBD2a isoform with antibodies that detect both MBD2a and MBD2b, suggesting that MBD2b is a rare form in this tissue (data not shown). The relative abundance of MBD2 in the colon is compatible with the hypothesis that various concentrations of this CpG methylation-dependent repressor contribute to the control of differential gene expression in colon and duodenum. To test this idea, we sought to determine whether expression of the ExPa genes is affected by artificial modulation of MBD2 concentrations in a model system: the human colon cancer cell line HCT116. We depleted MBD2 by stably expressing an shRNA directed against MBD2 mRNA. Real-time RT-PCR showed an mRNA knockdown of more than 90% in two shRNA-expressing clones (Fig. 4A), and Western blot analysis confirmed almost complete depletion of both MBD2a and MBD2b isoforms (Fig. 4B). This treatment caused >10-fold overexpression of two digestive enzymes, encoded by the genes TFF2 and CEL, in both MBD2-depleted cell clones (Fig. 4C). A more modest downregulation of MBD2 that paralleled the 5-fold difference between mouse duodenum and colon was achieved by transfecting siRNAs targeted against MBD2 mRNA (Fig. 4D and E) and once again led to >10-fold upregulation of TFF2 and CEL gene expression (Fig. 4F). Therefore, the modulation of MBD2 levels is sufficient to regulate the expression of these genes in a human colon cell line. We also tested the effects of DNA hypomethylation on these genes, using a DNMT1–/–, DNMT3b–/– double mutant derivative of HCT116 cells (28). The double-knockout (DKO) cells carry mutations in the DNA methyltransferases DNMT1 and DNMT3b and show reduced DNA methylation levels, as monitored by bisulfite sequencing (Fig. 5A). We detected 10-fold upregulation of TFF2 and P48 and 4-fold overexpression of CEL in DKO cells (Fig. 5B), indicating that DNA methylation, like MBD2, plays a role in silencing these genes.
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MBD2 binding and deacetylation of repressed exocrine pancreatic genes.
To test whether TFF2 is a direct MBD2 target, we carried out ChIP in wild-type, MBD2-depleted, and DNA methylation-deficient HCT116 cells. ChIP of genomic DNA corresponding to three primer pairs covering 1 kb upstream of the transcription start site was assayed by quantitative PCR. MBD2 was enriched at the TFF2 promoter in wild-type HCT116 cells, but binding to a domain extending 1 kb upstream of the transcription start site (Fig. 6A) was reduced by both MBD2-shRNA treatment and in DKO derivative cells (Fig. 6B). A similar reduction in MBD2 occupancy was observed at the P48 promoter in MBD2-deficient and DKO HCT116 cells (data not shown). Since MBD2 is known to recruit HDACs associated with the NuRD complex, we sought to determine whether reduced MBD2 binding was accompanied by an increase in histone acetylation levels at the TFF2 promoter. ChIP experiments revealed low levels of acetylated histone H4 in untreated HCT116 cells. These levels increased significantly after treatment with the histone deacetylase inhibitor trichostatin A but to a greater extent after MBD2 depletion by shRNA and in methylation-deficient DKO cells (Fig. 6C). Bisulfite genomic sequencing of the TFF2 promoter confirmed high levels of DNA methylation (>90%) in wild-type HCT116 cells but lower levels in DKO cells (Fig. 5A). The results demonstrate DNA methylation-dependent binding of MBD2 to the TFF2 promoter and show elevated histone acetylation in this region when MBD2 is depleted.
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DISCUSSION |
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Gene expression analysis along the length of the mouse intestine has shown that fewer specialized genes are expressed in the colon than in more proximal regions of the intestinal tract (4). Little is known about the molecular basis of segmental gene expression along the intestinal anterior-posterior axis. One hypothetical possibility is that expression patterns are regulated by the abundance of specific transcriptional activators in each segment. An alternative, but not mutually exclusive, possibility is that differential silencing of gene expression contributes to patterning through segmental variation in the abundance of transcriptional repressors. Previous work has indicated that variations in the expression of histone deacetylases, which are components of many corepressors, are crucial for embryonic development of the gastrointestinal tract (33). The observed anterior-posterior decline in transcriptional complexity along the intestine (4) could therefore be caused by increased cellular concentrations of broad specificity repressors in posterior intestinal segments.
We were initially prompted to study ExPa gene expression through an interest in the resistance of Mbd2-null mice to intestinal tumorigenesis in the Min mouse model (32). It is unlikely that our findings are relevant to this phenotype, however, for two major reasons. First, most adenomatous polyps in the Min mouse arise in the proximal intestinal tract (25), where we detected no altered expression of the ExPa genes. The location of ExPa gene mis-expression and the sites of tumorigenesis therefore do not match. Second, ExPa gene expression is not seen in all mice, whereas the tumor resistance phenotype appears fully penetrant. If failure to overexpress the ExPa genes abolished tumor resistance in mice, then a subset of animals (about half) with normal tumor susceptibility would have been detected. The absence of such a subset suggests that overexpression of ExPa genes is not causally related to tumor resistance. It is possible that other genes that are more subtly misregulated in the Mbd2-null intestine are relevant to the latter phenotype.
A puzzling feature of the colon phenotype is its incomplete penetrance. A continuum of ExPa gene expression levels is observed, ranging from very high (equivalent to duodenum) to no detectable expression in about half of the animals. It is common for transcriptional repressors to contribute to silencing as part of a redundant set of silencing mechanisms, and this can cause partial gene activation in a stochastic manner when one component is absent. Leakproof inactivation of genes on one X chromosome in female mammals, for example, relies on layered epigenetic silencing mechanisms, each of which reduces the probability per cell of gene reactivation (6). Indeed, MBD2 itself is not solely responsible for silencing of the interleukin-4 gene in TH cells, since not all MBD2-deficient TH cells express the gene (13). In spite of these precedents at the cellular level, variability of gene reactivation between members of an inbred population of mice is unexpected. Our studies make it unlikely that age, gender, or genetic background are critical factors affecting penetrance (see Results). Instead, they suggest that stochastic variation arises during development. We speculate that stem cells at the base of the colon crypts are variably dependent on MBD2 for silencing the ExPa genes in their differentiated descendants. MBD2 dependence or independence would be a stable feature that affects a particular crypt for the lifetime of its parent stem cell.
Mbd2-null animals that express ExPa genes showed coincident upregulation of p48. The p48 polypeptide is a component of Ptf1, a trimeric transcription factor that is essential for the development and maintenance of the differentiated state of the exocrine pancreas. A CpG island is located near the transcription start site of p48, which, like many CpG islands at highly tissue specifically expressed genes, is nonmethylated regardless of gene activity, except in cultured cell lines where aberrant methylation occurs. In view of its pivotal role in differentiation of the exocrine pancreas, we suspected that inappropriate p48 expression in the Mbd2-null colon is a trigger for ExPa gene expression. Under a simple hypothetical pathway, MBD2 would directly mediate repression of p48 in the colon. Although the p48 promoter is associated with a constitutively nonmethylated CpG island, which therefore should not bind MBD2, studies of the sequence requirements of p48 gene expression in mouse acinar and nonpancreatic cells have implicated a region extending up to 7 kb upstream of the p48 transcription start site in both positive and negative regulation of gene activity (18, 30). This region is not within the CpG island region and, accordingly, bisulfite sequence analysis in duodenum and colon detects CpG methylation. Also, MBD2 is bound to the equivalent region of human P48 by ChIP in HCT116 cells (data not shown). Taken together, our results are compatible with the hypothesis that the p48 gene is under direct regulatory control by MBD2.
MBD2 deficiency in HCT116 cells, which have silenced P48 via aberrant CpG island methylation, induced TFF2 and CEL, but not RIB1 or ELA2. Both RIB1 and ELA2 possess CpG-deficient promoters that do not correspond to CpG islands. The different behaviors of these genes in HCT116 cells and mouse colon could be explained if TFF2 and CEL are direct targets for MBD2 repression, whereas the induction of RIB1 and ELA2 requires the upstream expression of P48. In line with this hypothesis, the PTF1 transcriptional activator complex, which includes P48, has been shown to bind an enhancer of the ELA2 gene (29). Also, we show that MBD2 is associated with the TFF2 promoter region in HCT116 cells, and its absence is accompanied by alterations in the structure of TFF2 chromatin. Interestingly, a transgenic mouse with a reporter gene under the control of the multimerized Ela2 enhancer showed expression in the pancreas, duodenum and colon (29). It is conceivable that the absence of appropriate DNA methylation in the transgene prevented recruitment of MBD2, thereby permitting expression in the colon.
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ACKNOWLEDGMENTS |
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This study was supported by supported by Programme Grants from Cancer Research UK and the Wellcome Trust.
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FOOTNOTES |
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Published ahead of print on 12 March 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: CR-UK Beatson Institute for Cancer Research, Glasgow G6 11BD, United Kingdom.
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