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Cancer Research 68, 2708, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-6844
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

MUC1 Expression Is Regulated by DNA Methylation and Histone H3 Lysine 9 Modification in Cancer Cells

Norishige Yamada1, Yukari Nishida1, Hideaki Tsutsumida1, Tomofumi Hamada2, Masamichi Goto1, Michiyo Higashi1, Mitsuharu Nomoto1 and Suguru Yonezawa1

1 Department of Human Pathology, Field of Oncology and 2 Department of Oral and Maxillofacial Rehabilitation, Field of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Sakuragaoka, Kagoshima, Japan

Requests for reprints: Masamichi Goto, Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Phone: 81-99-275-5270; Fax: 81-99-265-7235; E-mail: masagoto{at}m2.kufm.kagoshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MUC1 is a transmembrane mucin that is highly expressed in various cancers and correlates with malignant potential. Important cancer-related genes such as p16 and E-cadherin are controlled epigenetically; however, MUC1 has been overlooked in epigenetics. Herein, we provide the first report that MUC1 gene expression is regulated by DNA methylation and histone H3 lysine 9 (H3-K9) modification of the MUC1 promoter. The recently developed MassARRAY assay was performed to investigate the DNA methylation status of 184 CpG sites from –2,753 to +263. Near the transcriptional start site, the DNA methylation level of MUC1-negative cancer cell lines (e.g., MDA-MB-453) was high, whereas that of MUC1-positive cell lines (e.g., MCF-7) was low. Histone H3-K9 modification status was also closely related to MUC1 gene expression. Furthermore, MUC1 mRNA expression in MUC1-negative cells was restored by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC1 gene expression, and this study defines MUC1 as a new member of the class of epigenetically controlled genes. An understanding of the epigenetic changes of MUC1 may be of importance for diagnosis of carcinogenic risk and prediction of outcome for cancer patients. [Cancer Res 2008;68(8):2708–16]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mucins are high molecular weight glycoproteins with oligosaccharides attached to serine or threonine residues of the mucin core protein backbone via O-glycosidic linkages. These proteins are produced by various epithelial cells. The MUC1 transmembrane glycoprotein is a member of the mucin family that is expressed at a basal level by normal ductal epithelial cells of secretory organs, including pancreas, breast, lung, and gastrointestinal tract (1), and is overexpressed and aberrantly glycosylated in most cases of adenocarcinoma (2). An elevated level of MUC1 protein plays a role in tumor progression, especially in the process of metastasis (3, 4). Our immunohistochemical studies of mucin expression in various human tumors, including pancreatic tumors, have also shown that MUC1 expression is related to the invasive proliferation of tumors and is predictive of a poor outcome for patients (5).

Regulation of MUC1 expression has been studied extensively, and TATA box, ubiquitous cis-acting elements, GC-boxes, and binding sites for transcriptional regulators such as Sp1 and GATA3 have been identified in the MUC1 promoter (6–8). However, the methylation status of the large number of CpG sites in the promoter region has yet to be clarified, in contrast to the MUC1 coding region (9). Methylation of cytosine in genomic DNA plays an important role in gene regulation and especially in gene silencing (10, 11), and generally, the promoter region of a transcribed gene is hypomethylated (12, 13). To examine the methylation profiles of 184 CpG sites in ~3,000 bp in the MUC1 promoter in cancer cell lines, we performed a methylation analysis (MassARRAY) that uses base-specific cleavage of nucleic acids (14). Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This method permits high-throughput identification of methylation sites and semiquantitative measurement at single or multiple CpG sites. Based on the results of the MassARRAY analysis, we designed methylation-specific PCR (MSP) primers to ensure the CpG sites related to gene expression.

Modification of histone tails also plays a critical role in epigenetic silencing (15, 16). Acetylation of lysine residues on histone H3 leads to formation of an open chromatin structure, whereas methylation of K9 on histone H3 is a marker of heterochromatin (17). Functional interactions among DNA methylation, histone modification, and gene expression is the focus of studies on epigenetic control (18, 19). Therefore, to reveal the relationship between DNA methylation and histone modification, chromatin immunoprecipitation (ChIP) primers were designed to target regions similar to those targeted by the MSP primers. We also treated cells that were MUC1-negative or those with low expression of MUC1 with a DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AzadC), and a histone deacetylase inhibitor, trichostatin A, to confirm that DNA methylation and histone H3 modification suppress expression of MUC1 mRNA. In this report, we describe an epigenetic mechanism through which MUC1 gene expression is regulated by a tightly related combination of DNA methylation and histone histone H3 lysine 9 (H3-K9) modification in the 5' flanking region of the MUC1 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and treatment. Human pancreatic carcinoma cell lines HPAFII (MUC1+), BxPC3 (MUC1+), PANC1 (MUC1+/–), human breast cancer cell lines MCF-7 (MUC1+), T-47D (MUC1+), MDA-MB-453 (MUC1–) and human colon adenocarcinoma cell lines Caco2 (MUC1–), and LS174T (MUC1+/–) were obtained from American Type Culture Collection. HPAFII, MCF-7, LS174T, and Caco2 cells were cultured in Eagle's MEM (Sigma), PANC1 cells were cultured in DMEM (Sigma), BxPC3 and T-47D cells were cultured in RPMI 1640 (Sigma), and MDA-MB-453 cells were cultured in Leibovitz's L-15 medium (Invitrogen). All media were supplemented with 10% fetal bovine serum (Invitrogen) and 100 U/mL penicillin/100 µg/mL streptomycin (Sigma). MUC1-negative cells or cells with low MUC1 were split 24 h before treatment. PANC1, MDA-MB-453, and LS174T cells were incubated for 5 d with 100 µmol/L 5-AzadC (Sigma) and/or for 5 d with 500 nmol/L trichostatin A (TSA; Sigma). Caco2 cells were incubated for 5 d with 100 µmol/L 5-AzadC and/or for 5 d with 50 nmol/L TSA. Media were changed every 24 h.

Quantitative reverse transcription-PCR analysis. The mRNA from cells that had or had not undergone 5-AzadC, TSA, or 5-AzadC/TSA combination treatment was purified with a RNeasy Mini kit (Qiagen). Of a total of 100 µL mRNA, 20 µL was reverse transcribed with Random-Hexamers (Applied Biosystems). A 3.5-µL cDNA aliquot was amplified in 25 µL of 2x Taq Man Universal Master Mix, 2.5 µL of 20xTarget Assay Mix, and 2.5 µL of 20x Control Assay Mix (Applied Biosystems) under the following PCR conditions: 2 min at 50°C, 10 min at 95°C, 45 cycles of 15 s at 95°C, and 1 min at 60°C. The primers and probes were designed and synthesized by Applied Biosystems. The product number of the Target Assay Mix used for MUC1 was Hs00410317. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; product number 4310884E) was used to calibrate the original concentration of mRNA; i.e., the concentration of mRNA in the cell was defined as the ratio of target mRNA copies versus GAPDH mRNA copies. In this analysis, data from three separate experiments were averaged.

Immunohistochemical staining. MUC1 protein expression levels were assessed by immunohistochemistry. MUC1 was detected using a monoclonal antibody against MUC1 core glycoprotein (mouse IgG; Novocastra Laboratories Ltd.; dilution, 1:100 for cell culture; incubation period, 1 h at 37°C). Immunohistochemical staining was performed by an immunoperoxidase method using a Vectastain Elite ABC kit (Vector Laboratories), as described previously (20).

MUC1 gene promoter sequencing. Genomic DNA was extracted from the seven cell lines using a DNeasy Tissue System (Qiagen) according to the manufacturer's instructions. DNA was PCR amplified using six pairs of sense and antisense primers (Table 1 ) in the full-length MUC1 promoter. Primer sequences were based on a previously published sequence (21). PCR fragments were sequenced using single strand sequencing method (Hokkaido System Science Co., Ltd.). Sequences were analyzed with an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems).


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  		Table 1. Synthetic oligonucleotides used in the study

 
Quantitative methylation analysis. Quantitative methylation analysis of the MUC1 promoter was performed using the MassARRAY Compact system (Hitachi High-Technologies Corporation; ref. 14). DNA from cell lines was extracted using a DNeasy Tissue System (Qiagen). A 1-mg sample of DNA was converted with sodium bisulfite using an EZ DNA methylation kit (Zymo Research) and the modified DNA was amplified by PCR. The target regions were amplified using the primer pairs shown in Table 1. Each forward primer is tagged with a 10 mer (5'-AGG AAG AGA G-3') to balance the PCR and a reverse primer (5'-CAG TAA TAC GAC TCA CTA TAG GGA GAA GGC T-3') with a T7-promoter tag for in vitro transcription. PCR amplification was performed with the following variables: hot start at 94°C for 15 min, followed by denaturing at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 1 min for 45 cycles, and final incubation at 72°C for 3 min. Unincorporated deoxynucleotide triphosphates were dephosphorylated by adding 2 µL of premix including 0.3 units Shrimp Alkaline Phosphate (SAP; Sequenom). The reaction mixture was incubated at 37°C for 40 min, and SAP was then heat inactivated for 5 min at 85°C. After SAP treatment, 2 µL of the PCR products were used as a template for in vitro transcription, and RNase A cleavage was used for the reverse reaction, following the manufacturer's instructions (Sequenom). The samples were conditioned and spotted on a 384-pad Spectro-CHIP (Sequenom) using a MassARRAY nanodispenser (Samsung), followed by spectral acquisition on a MassARRAY Analyzer Compact MALDI-TOF MS (Sequenom). The resultant methylation calls were analyzed with EpiTyper software v1.0 (Sequenom) to generate quantitative results for each CpG site or an aggregate of multiple CpG sites.

DNA extraction and DNA MSP analysis. DNA from cell lines was extracted using a DNeasy Tissue System (Qiagen), according to the manufacturer's instructions. Bisulfite modification of the genomic DNA was carried out using a Epitect Bisulfite kit (Qiagen), and the modified DNA was amplified by PCR using a Fast Cycling PCR kit (Qiagen). The target regions were amplified using the primer pairs shown in Table 1. The PCR conditions were 95°C for 5 min, 36 cycles at 96°C for 5 s, 58°C for 5 s, and 68°C for 3 s, with a final extension reaction at 72°C for 1 min. The amplified products were subjected to 1.5% agarose gel electrophoresis.

ChIP assay. The ChIP assay was carried out using an EpiQuik Chromatin Immunoprecipitation kit (Epigentek, Inc.), according to the manufacturer's instructions. The nucleoprotein complexes were sonicated to reduce the sizes of DNA fragments to 300 to 500 bp using a Bioruptor (Cosmo Bio). Four micorgrams of normal mouse IgG was used as the negative control, and antidimethyl histone H3-K9 antibody (Abcam) and antiacetyl histone H3-K9 antibody (Upstate Biotechnologies) were used for each immunoprecipitation. Immunoprecipitated DNA was amplified by PCR using a Fast Cycling PCR kit (Qiagen). The ChIP primers were designed to target a region similar to the target region of the MSP primers (Table 1). The PCR conditions were 95°C for 5 min, 38 to 40 cycles at 96°C for 5 s, 59°C for 5 s, and 68°C for 7 s, with a final extension reaction at 72°C for 1 min. The amplified products were subjected to 1.5% agarose gel electrophoresis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Restoration of the MUC1 mRNA level by 5-AzadC and 5-AzadC/TSA. Experiments were performed using three human pancreatic cancer cell lines, HPAFII, BxPC-3, and PANC1; three human breast cancer cell lines, MCF-7, T-47D, and MDA-MB-453; and two human colon adenocarcinoma cell lines, Caco2, and LS174T. Expression levels of MUC1 mRNA and protein in the cell lines were examined by reverse transcription-PCR (RT-PCR) analysis and immunohistochemical staining (Fig. 1A and B ). HPAFII, BxPC-3, MCF-7, and T-47D cells expressed MUC1, but PANC1, MDA-MB-453, Caco2, and LS174T cells did not do so. To examine the effects of DNA methylation and histone modification on MUC1 gene expression, quantitative RT-PCR analysis was performed in MUC1-negative cells or cells with low expression of MUC1 treated with a DNA demethylating agent, 5-AzadC, a histone deacetylase inhibitor, TSA, or 5-AzadC/TSA in combination (Fig. 1C). A 3-fold recovery of MUC1 mRNA expression was observed after 5-AzadC or 5-AzadC/TSA treatment of PANC1 cells, but there was no effect of TSA alone. In MDA-MB-453 cells, treatment with 5-AzadC or 5-AzadC/TSA resulted in a 100-fold increase in expression of MUC1 mRNA compared with controls, whereas TSA alone gave a 3-fold restoration. Treatment of Caco2 cells with 5-AzadC or 5-AzadC/TSA showed an increase of 10- to 25-fold in MUC1 mRNA, whereas the level remained constant after treatment with TSA alone. Therefore, in these three cell lines, treatment with 5-AzadC or 5-AzadC/TSA significantly restored the MUC1 mRNA level, compared with treatment with TSA alone. In contrast, LS174T cells showed restoration of MUC1 expression only when treated with 5-AzadC/TSA. These results suggest that a combination of DNA methylation and histone modification may affect expression of the MUC1 gene.


Figure 1
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  		Figure 1. Expression of the MUC1 gene examined by quantitative RT-PCR and immunohistochemistry. A, quantitative RT-PCR results in eight cancer cell lines. Bars, gene expression levels relative to those in PANC1 cells. HPAFII, BxPC-3, MCF-7, and T-47D cells showed high expression of MUC1 mRNA, whereas PANC1, MDA-MB-453, Caco2, and LS174T cells had no or low expression. B, MUC1 immunoreactivity in eight cancer cell lines. MUC1 expression was consistent with the results of RT-PCR. C, quantitative RT-PCR results before and after treatment with 5-AzadC, TSA, and 5-AzadC/TSA in combination in cells with little or no MUC1 expression. After 5-AzadC and 5-AzadC/TSA treatment, PANC1, MDA-MB-453, and Caco2 cells showed significant restoration of MUC1 expression. In LS174T cells, MUC1 mRNA was restored only when treated with 5-AzadC/TSA.

 
Next, to rule out the contribution of single nucleotide polymorphisms (SNP) in control of MUC1 expression, we sequenced a 2.8-kb promoter region of the human MUC1 gene from seven cell lines (Fig. 2 ). Sixty bases differed from the previously published MUC1 promoter sequence (21), but all the sequences in the seven cell lines were identical. Additionally, our sequence showed a 99% match with the sequences with Genbank accession numbers NT_004487.18 and NW_925683.1.


Figure 2
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  		Figure 2. The human MUC1 gene promoter sequence, which spans positions –2,753 to +263 with respect to the transcription start site. The numbers of CpG sites and the transcriptional start site +1 (arrow) are shown.

 
Quantification of DNA methylation in the MUC1 gene promoter. To map methylated cytosines in the MUC1 promoter in eight cancer cell lines, we used quantitative methylation analysis using the MassARRAY Compact system and mapped the efficacious data (Fig. 3 ). This approach has been proven to be a powerful DNA methylation analysis tool (14). Near the transcriptional start site (~–100 to +100), a high level of CpG methylation was observed in MUC1-negative cell lines and those with low MUC1 expression (PANC1, MDA-MB-453, and Caco2) compared with MUC1-positive cell lines (HPAFII, BxPC-3, MCF-7, and T-47D). However, LS174T cells, which have low MUC1 expression, showed hypomethylation of the MUC1 promoter. The methylation status of CpG sites 1 to 173 did not influence expression of the MUC1 gene. These results indicate that hypomethylation of the 5' flanking region of the promoter is linked to MUC1 expression, except in LS174T cells.


Figure 3
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  		Figure 3. Quantitative methylation analysis of CpG sites located in the MUC1 promoter using a MassARRAY Compact system. The efficacious data was mapped and different colors display relative methylation changes in 10% increments (green, 0%; red, 100% methylated). *, MUC1-positive cell lines. Of the cells with little or no MUC1 expression, PANC1, MDA-MB-453, and Caco2 cells showed high CpG methylation, but LS174T cells showed CpG hypomethylation in the MUC1 promoter. The methylation level in MUC1-positive HPAFII, BxPC-3, MCF-7, and T-47D cells was low.

 
MSP analysis of the MUC1 gene promoter in eight cancer cell lines. To confirm the MassARRAY results, primers for MSP were designed to target the 5' flanking region of the MUC1 promoter (Fig. 4A ; Table 1). Four CpG sites (Nos. 177, 178, 179, and 181) were included in the MSP primer. These sites showed 0% to 4%, 2% to 21%, 1% to 17%, and 2% to 26% methylation, respectively, in MUC1-positive cells, and 29% to 63%, 66% to 86%, 53% to 67%, and 79% to 93% methylation, respectively, in all MUC1-negative/low cells except for LS174T cells. The LS174T cells showed 1%, 5%, 4%, and 3% methylation at the respective sites. To ensure the reliability of the MSP primer, MSP analysis was performed on the eight cell lines (Fig. 4B). An unmethylated band (lanes indicated by U; Fig. 4B) was clearly obtained in MUC1-positive HPAFII, BxPC-3, MCF-7, and T-47D cells, whereas a methylation band (lanes indicated by M; Fig. 4B) was observed in PANC1, MDA-MB-453, and Caco2 cells. Overall, the MSP results were consistent with those from MassARRAY analysis and showed the methylation status of the MUC1 promoter in the eight cell lines.


Figure 4
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  		Figure 4. MSP and ChIP analyses in the MUC1 promoter region. A, schematic representation of the MUC1 gene promoter region, which spans positions –2,753 to +263 with respect to the transcription start site. There are 184 CpG sites, and the MSP and ChIP primers used in the study are indicated. B, MSP analysis of the MUC1 gene promoter region in eight cell lines. The PCR-products labeled M (methylated) were generated by methylation-specific primers, and those labeled U (unmethylated) were generated by primers specific for unmethylated DNA. The methylated allele was detected in PANC1, MDA-MB-453, and Caco2 cells, whereas the unmethylated allele was detected in HPAFII, BxPC-3, MCF-7, T-47D, and LS174T cells. *, MUC1-positive cell lines. C, ChIP analysis of histone H3-K9 modification in the MUC1 promoter region (ChIP regions 1–3) in eight cancer cell lines. Reactions were performed by PCR using input DNA. A negative control reaction using an aliquot precipitated with negative control normal mouse IgG antibody (Nc) is also shown. Unbound and bound fractions in each ChIP experiment are shown. In ChIP regions 1 to 3, histone H3-K9 dimethylation was observed in cells with little or no MUC1 expression, except for PANC1 cells, which showed high acetylation of histone H3-K9. Acetylation of histone H3-K9 was detected in all MUC1-positive cells. *, MUC1-positive cell lines.

 
Correlation between CpG methylation and histone H3-K9 modification in the MUC1 promoter. Three ChIP primers were designed to examine the relationship between DNA methylation and histone modification. ChIP3 primer was designed to target regions similar to those targeted by the MSP primer (Fig. 4A; Table 1). To determine the profile of histone H3-K9 modification in MUC1-positive and MUC1-negative/low cells, ChIP assays were performed for three regions using antidimethyl H3-K9 and antiacetyl H3-K9 antibodies. Methylation of lysine 9 of histone H3 (H3-meK9) facilitates formation of heterochromatin, and elevated levels of H3-meK9 at promoter sequences are associated with suppression of gene expression (22, 23). In MUC1-negative or cells with low MUC1 expression (MDA-MB-453, Caco2, and LS174T), dimethylation of H3-K9 was found in all regions (ChIP1–3; Fig. 4C). Acetylation of lysine 9 of histone H3 is associated with euchromatin formation, and acetylation of promoter-proximal histones is associated with gene expression (24). The acetylation level of H3-K9 in MUC1-positive cells (HPAFII, BxPC-3, MCF-7, and T-47D) was high in the MUC1 promoter (Fig. 4C). In PANC1 cells (low MUC1 expression) high acetylation of H3-K9 was observed. Collectively, MSP and ChIP analysis revealed that DNA demethylation, histone H3-K9 demethylation, and histone H3-K9 acetylation in the 5' flanking region of the MUC1 promoter may all be necessary for MUC1 gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulation of human MUC1 gene expression has been studied intensively, and SP1, tumor necrosis factor, INF,and GATA3 have been reported to activate MUC1 transcription (7, 25–27). However, the methylation status of CpG islands existing in the MUC1 promoter has yet to be elucidated, and our data revealed the details of DNA methylation and histone H3-K9 modification on the full-length MUC1 promoter in MUC1-positive and MUC1-negative cancer cell lines for the first time. Before beginning the epigenetic analysis, we performed sequencing of the MUC1 promoter in seven cancer cells to examine possible regulatory SNPs (rSNP). rSNPs are usually promoter region mutations that cause variation in gene expression levels (28). Our results were perfectly congruent in the seven cell lines and largely matched the data for Genbank accession numbers NT_004487.18 and NW_925683.1, although some bases differed from the published MUC1 promoter sequence (21). We found no correlation between rSNPs and MUC1 expression, and therefore, we examined the DNA methylation status of the MUC1 promoter in eight cancer cell lines.

Methylation of cytosine residues at CpG dinucleotides is an important epigenetic change that is linked to transcriptional repression and regulation of chromatin structure (29). There are several techniques for evaluation of CpG methylation, but most can only analyze a restricted set of CpG sites in a target region. Thus, we used the MassARRAY method to overcome this limitation. In eight cancer cell lines, methylation of CpG sites 174 to 182 was inversely correlated with MUC1 gene expression, whereas methylation of sites 1 to 173 was almost unrelated to gene expression (Fig. 3). Gonzalgo et al. (30) suggested that a relatively low number of methylation errors may be sufficient to initiate a reduction of gene expression. Our results are in agreement with this hypothesis because we observed a high level of CpG methylation in MUC1-negative/low-expression cell lines only in the vicinity of the transcriptional start site. To validate the detailed methylation status in the 5' flanking region of the MUC1 promoter, we selected four CpG sites (177, 178, 179, and 181), at which methylation may affect MUC1 gene expression, and designed a corresponding MSP primer. Our MSP results were consistent with the MassARRAY data in all cancer cell lines. Interestingly, despite LS174T cells showing low expression of MUC1, the MUC1 promoter was mostly unmethylated in these cells.

To examine the possibility of another form of epigenetic control for the MUC1 gene, we investigated histone modification in the eight cancer cell lines. Similarly to CpG methylation, it has become increasingly evident that histone modification can contribute to gene regulation (31). To examine the relationship between DNA methylation and histone modification in MUC1 expression, a ChIP primer was designed to target regions similar to those targeted by the MSP primer (Fig. 4A). Nguyen et al. (22) showed that aberrantly silenced genes in cancer cells exhibit a heterochromatic structure that is characterized by H3-K9 hypermethylation. In contrast, histone H3 acetylated at lysine 9 in a gene promoter region is associated with low nucleosome density near the transcription start site in human cells (32). Based on these findings, we performed a ChIP assay using antidimethyl H3-K9 and antiacetyl H3-K9 antibodies. The presence of dimethyl H3-K9 in MUC1-negative/low cells was confirmed in ChIP regions 1 to 3, including in LS174T cells (Fig. 4C). In LS174T cells, heterochromatin structure induced by H3-K9 methylation may down-regulate MUC1 expression. Histone H3-K9 was more highly acetylated in MUC1-positive cells than in MUC1-negative/low cells. Unexpectedly, PANC1 cells (low MUC1 expression) also showed a high degree of H3-K9 acetylation in all ChIP primer regions. As contrasted with LS174T cells, methylated CpGs in the 5' flanking region of the MUC1 promoter may be related to suppression of MUC1 gene transcription in PANC1 cells.

To investigate the combined role of DNA methylation and histone modification in the regulation of MUC1 gene expression, we also treated MUC1-negative or low-expression cancer cell lines with 5-AzadC and/or TSA (Fig. 1C). Kondo et al. (33) showed that 5-AzadC or a combination of 5-AzadC and TSA, but not TSA alone, reactivates tumor suppressor gene expression at silenced loci (e.g., p16). Our results in MDA-MB-453 and Caco2 cell lines are in good agreement with this observation because we showed that treatment with 5-AzadC or 5-AzadC/TSA significantly restored the MUC1 mRNA level, compared with TSA alone. However, treatment with TSA did not restore the MUC1 mRNA level in PANC1 cells (Fig. 1C), raising the possibility that euchromatin formation remains unchanged due to the acetylated histone H3-K9 (Fig. 4C), and 5-AzadC did not restore the MUC1 mRNA level in LS174T cells (Fig. 1C), which implies that 5-AzadC alone has no effect on an already hypomethylated MUC1 gene promoter (Fig. 3).

From these results, we propose four patterns of epigenetic control of MUC1 gene expression, as shown in Fig. 5 . First, DNA demethylation, histone H3-K9 demethylation and histone H3-K9 acetylation in the 5' flanking region of the MUC1 promoter are all necessary in cancer cells with high MUC1 expression (HPAF II, BxPC-3, MCF-7, and T-47D; Fig. 5A). Second, in cases of histone H3-K9 demethylation and histone H3-K9 acetylation, MUC1 can be expressed slightly, even if CpG is methylated (PANC1; Fig. 5B). Third, with methylated histone H3-K9, MUC1 expression is reduced regardless of CpG demethylation (LS174T; Fig. 5C). Fourth, methylation of both DNA and histone H3-K9 results in suppression of MUC1 expression in cells such as MDA-MB-453 and Caco2 (Fig. 5D). Taken together, these results show that neither DNA methylation nor histone modification alone fully determine the expression of MUC1, suggesting that expression is regulated by a combination of DNA methylation and histone H3-K9 modification in the 5' flanking region of the MUC1 promoter. An understanding of these intimately correlated epigenetic changes for the MUC1 gene may be of importance for diagnosis of carcinogenic risk and prediction of outcomes of patients.


Figure 5
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  		Figure 5. A model depicting changes in chromatin modification and DNA methylation of the MUC1 gene promoter region in eight cancer cell lines. Four mechanisms of epigenetic control of MUC1 expression are proposed. Patterns A and B indicate euchromatin states, whereas patterns C and D show heterochromatin formation. The core region is indicated by brackets. {circ}, unmethylated CpG sites; bullet, methylated CpG sites. HPAFII, BxPC-3, MCF-7, and T-47D cells, in which MUC1 is overexpressed, follow pattern A. PANC1 and LS174T cells follow patterns B and C, respectively, and both express a low level of MUC1. MUC1-negative MDA-MB-453 and Caco2 cells follow pattern D.

 

   Acknowledgments
 
Grant support: Scientific Research on Priority Areas 18014024 from the Ministry of Education, Science, Sports, Culture and Technology, Japan (S. Yonezawa) and the Kodama Memorial Foundation (N. Yamada).

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 Yoshiko Arimura and Yukari Nishimura for their excellent technical assistance, and Hiromichi Iai (Hitachi High-Technologies Corporation, Tokyo, Japan) and Katsuhiko Hashimitsu (Hitachi High-Tech Manufacturing & Service Corporation, Ibaraki, Japan) for their help with quantitative DNA methylation analysis.

Conflicts of interest: The authors have no conflicts of interest.

Received 12/26/07. Accepted 1/28/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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N. RABIAU, P. DECHELOTTE, L. GUY, S. SATIH, R. BOSVIEL, L. FONTANA, J.-L. KEMENY, J.-P. BOITEUX, Y.-J. BIGNON, and D. BERNARD-GALLON
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