Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Cancer Health Disparities Conference 2009
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[Cancer Research 63, 498-504, January 15, 2003]
© 2003 American Association for Cancer Research

Tumor Biology

Methyl-CpG-binding Domain Protein-2 Mediates Transcriptional Repression Associated with Hypermethylated GSTP1 CpG Islands in MCF-7 Breast Cancer Cells1

Xiaohui Lin and William G. Nelson2

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 21231


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GSTP1, encoding the {pi}-class glutathione S-transferase, is commonly inactivated by somatic CpGisland hypermethylation in cancers of the prostate, liver, and breast. We report here thathypermethylation of CpG dinucleotides at the 5' transcriptional regulatory region was sufficient to inhibit GSTP1 transcription in MCF-7 breast cancer cells and that repression of GSTP1 transcription was mediated in part by the methyl-CpG-binding domain (MBD) protein MBD2. MCF-7 breast cancer cells contained only hypermethylated GSTP1 CpG island alleles and failed to express GSTP1 mRNA or GSTP1 polypeptides. In contrast, MCF-7/ADR cells contained only unmethylated GSTP1 CpG island alleles and exhibited abundant GSTP1 expression. Chromatin immunoprecipitation analysis detected the presence of MBD2 and DNMT1 at the GSTP1 promoter in MCF-7 breast cancer cells but not in MCF-7/ADR breast cancer cells. In a test of the contribution of MBD2 to GSTP1 repression in MCF-7 breast cancer cells, transfection of small interference RNA complementary to MBD2 mRNA into MCF-7 cells both reduced MBD2 polypeptide levels and stimulated GSTP1 mRNA expression. These findings implicate MBD2 in GSTP1 silencing associated with somatic GSTP1 CpG island hypermethylation in breast cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Somatic changes in CpG dinucleotide methylation patterns occur commonly during the pathogenesis of human cancers (1, 2, 3) . CpG dinucleotides clustered into CpG islands near transcriptional regulatory regions of most genes are typically not methylated in normal cells (4 , 5) . In cancer cells, hypermethylation of such CpG island sequences has been associated with transcriptional silencing of many critical cancer genes (1, 2, 3) . For some genes, CpG island hypermethylation may inhibit transcription by interfering with the binding and/or function of transcriptional trans-activators; for other genes, hypermethylation of CpG dinucleotides near the transcriptional regulatory region appears to recruit MBD3 family proteins capable of mediating transcriptional repression via effects on chromatin structure (6 , 7) .

GSTP1, encoding the {pi}-class glutathione S-transferase, has been reported to be the target of somatic CpG island hypermethylation in >90% of prostate cancers (8, 9, 10, 11) , >80% of liver cancers (12) , and >30% of breast cancers (13) . For each of these cancer cell types, GSTP1CpG island hypermethylation appears likely to be responsible for absence of GSTP1 expression. Treatment of LNCaP prostate cancer cells, Hep3B liver cancer cells, and MCF-7 breast cancer cells with inhibitors of DNMTs leads to reductions in GSTP1 CpG island methylation and increases in GSTP1 mRNA and GSTP1 polypeptide expression (8 , 12 , 14, 15, 16) . Furthermore, SssI CpG methylase-catalyzed methylation of GSTP1 transcriptional regulatory sequences has been found to decrease GSTP1 promoter activity on transfection into LNCaP prostate cancer cells (8 , 15) and K562 erythroleukemia cells (17) . The MBD family protein MBD2 present in nuclear extracts prepared from LNCaP prostate cancer cells appears able to bind SssI-treated GSTP1 promoter sequences in electrophoretic mobility shift experiments (15) . However, whether MBD2, or any other MBD family protein, binds hypermethylated GSTP1 CpG island sequences in cancer cells to repress GSTP1 transcription has not been tested.

Although MCF-7 breast cancer cells contain only hypermethylated GSTP1 CpG island alleles and are devoid of GSTP1 mRNA (14) , variant MCF-7 breast cancer cell lines selected for resistance to doxorubicin and other antineoplastic drugs express high levels of GSTP1 mRNA and GSTP1 polypeptides (18, 19, 20) . In fact, GSTP1 cDNA was first cloned from an antineoplastic drug-resistant variant MCF-7 breast cancer cell line (19) . The differential GSTP1 expression in GSTP– MCF-7 breast cancer cells versus GSTP+ breast cancer cells has been attributed to differences in GSTP1 mRNA stability (21 , 22) , differences in sequence-specific trans-repressor activity, targeting a region from -105 to -86 5' of the transcriptional start site in the GSTP1 promoter (23 , 24) , and differences in GSTP1 CpG island methylation (14) . However, the relative contribution of GSTP1 CpG island hypermethylation to the differences in GSTP1 expression between MCF-7 breast cancer cells and antineoplastic drug-resistant variant MCF-7 breast cancer cells has not been established. Furthermore, the mechanism by which GSTP1 CpG island hypermethylation prevents GSTP1 mRNA expression in MCF-7 breast cancer cells has not been elucidated. We report here the results of a study of GSTP1 CpG island hypermethylation and GSTP1 regulation in MCF-7 breast cancer cells and antineoplastic drug-resistant MCF-7/ADR breast cancer cells (25 , 26) . The data collected suggest that GSTP1 is silenced in MCF-7 breast cancer cells as a result of CpG island hypermethylation and that the repression of hypermethylated GSTP1 CpG island alleles is at least partly mediated by MBD2.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
GSTP1 Expression in MCF-7 and MCF-7/ADR Breast Cancer Cells Treated with 5-aza-C.
MCF-7 and MCF-7/ADR breast cancer cells, obtained from Nancy E. Davidson (Johns Hopkins), were propagated in vitro in MEM growth medium with 10% calf serum (19 , 20) . Immunoblot analysis for GSTP1 polypeptides, Northern blot analysis for GSTP1 and estrogen receptor mRNA, nuclear run-on transcription analysis, and Southern blot analysis using 32-P-labeled GSTP1 cDNA were all accomplished as described previously (8 , 10 , 27 , 28) . To assess the effects of DNMT and/or HDAC inhibition on GSTP1 expression in MCF-7 breast cancer cells, the cells were treated with 5-aza-C (Sigma Chemicals Co.) or with TSA (Sigma) for <=72 h in complete growth medium. To generate stable MCF-7–5-aza-C cells, growing MCF-7 cells were continuously exposed to 10 mM 5-aza-C for >6 months. MCF-7–5-aza-C subclones were isolated by limiting dilution cloning and maintained in complete growth medium containing 5-aza-C.

Assessment of GSTP1 CpG Island Methylation by Southern Blot Analysis and Bisulfite Genomic Sequencing.
To detect GSTP1 CpG island hypermethylation in MCF-7 cells, MCF-7/ADR cells, and MCF-7–5-aza-C subclones, Southern blot analysis of genomic DNA, after treatment with HindIII, EcoRI, and the 5-mCpG-sensitive endonuclease NotI, was performed using 32P-labeled GSTP1 cDNA probes as described previously (10) . 5-mCpG dinucleotides were mapped at individual GSTP1 CpG island alleles using a bisulfite genomic sequencing approach reported in detail previously (8) . Genomic DNA was digested with EcoRI, treated with bisulfite, and then subjected to two rounds of PCR targeting GSTP1 CpG island sequences (first PCR reaction primers: GenBank positions -636 to -613, 5'-ACA/GCAACCTATAATTCCACCTACTC-3', and +117 to +94, 5'-GTT/CGGGAGTTGGGGTTTGATGTTG-3'; second PCR reaction primers: GenBank positions -535 to -512, 5'-AACCTAAACCACAACA/GTAAAACAT-3', and +89 to +66, 5'-TTGGTTTTATGTTGGGAGTTTTGA-3'). The resultant PCR products were purified by separation on agarose gels (Life Technologies, Inc.), recovered from the agarose (using a QIAquick gel extraction kit, Qiagen), and then cloned by ligation into pCR 2.1pTOPO cloning vectors (using a TOPO kit; Invitrogen). Cloned plasmid DNAs, representing individual GSTP1 CpG island alleles, were subjected to DNA sequence analysis using a cycle sequencing approach with M13 sequencing primers, dye-labeled terminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit; Perkin-Elmer), and an ABI automated sequencer.

ChIP Targeting GSTP1 Promoter.
MCF-7 and MCF-7/ADR cells were fixed with 1% formalin for 10 min at 37°C, treated with a lysis buffer [1% SDS and 10 mM EDTA in 50 mM Tris-HCl (pH 8.1)] to liberate nucleoprotein complexes, and then sonicated to reduce DNA fragment size to 400–500 bp. The complexes were then immunoprecipitated with antibodies specific for acetylated H4, MeCP2, MBD2/3, and DNMT1 (Upstate Biotechnology; Ref. 29 ). The amount of GSTP1 promoter DNA recovered with the precipitated complexes was estimated by quantitative PCR [using a QuantiTect SYBR Green PCR kit, an iCycler iQ multicolor real time PCR system (Bio-Rad), and primers 5'-GGGACCCTCCAGAAGAGC-3' and 5'-ACTCACTGGTGGCGAAGACT-3']. MDR1 promoter DNA recovered with precipitated nucleoprotein complexes was also detected by quantitative PCR (primers 5'-CCTCCTGGAAATTCAACCTG-3' and 5'-TGTGGCAAAGAGAGCGAAG-3').

siRNA "Knock-Down" of MBD2, MBD3, MeCP2, and DMNT1 Expression.
siRNAs (30) , capable of targeting mRNAs encoding MBD2 (position 250–270, accession no. NM_003927), MBD3 (position 111–130, accession no. NM_003926), MeCP2 (position 291–311, accession no. NM_004992), DNMT1 (position 123–153, accession no. NM_001379), and lamin A (position 613–633, accession no. X03444), were synthesized by Xeragon or by Pharmacon Research. Around 105 MCF-7 cells were transfected (using Oligfectamine; Life Technologies, Inc.) with specific siRNAs. To knock-down protein expression in the highest fraction of cells, the siRNA transfection was repeated 72 h later. Knock-down efficiency was monitored by immunoblot analyses using antibodies specific for MDB2/3, MeCP2, DNMT1, and lamin A/C (Upstate Biotechnology; Ref. 29 ). Neither antibodies specific for MBD3 (Santa Cruz Biotechnology) or MDB2/3 (Upstate Biotechnology) were able to detect MBD3 polypeptides in MCF-7 cell protein extracts by immunoblot analysis.

Assessment of GSTP1 Promoter Function.
Growing MCF-7 cells were transfected with pCMV-ß-Gal (Promega), pGL3-Control (containing SV40 promoter sequences; Promega), pGL3-GSTP-1 (prepared by cloning GSTP1 promoter sequences, GenBank positions -408 to +36, into pGL3-Basic; Promega), pGL3-GSTP-1 treated with SssI CpG methylase (New England BioLabs), or pGL3-GSTP-6 (prepared by ligating GSTP1 promoter sequences, GenBank positions -65 to +36, into pGL3-Basic) in a manner described previously (8 , 16) . The transfected cells were then treated for 24 h with and without TSA (100 ng/ml) in complete growth medium. To assess reporter gene expression, the transfected cells were lysed and then assayed for ß-galactosidase activity (using the ß-Galactosidase Enzyme Assay System with Reporter Lysis Buffer; Promega) or for luciferase activity (using the Luciferase Assay System with Reporter Lysis Buffer; Promega). GSTP1 promoter activity in MCF-7 cells was also evaluated using a series of unmethylated GSTP1 promoter/CAT reporter constructs as described previously (23 , 24) . CAT reporter expression was assessed 48 h after transfection using an enzyme activity assay (FLASH CAT Nonradioactive Assay kit; Stratagene). The plasmids pCAT-Control (Promega) and pCMV-ß-gal (Stratagene) served as controls for transient transfection analyses.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To assess GSTP1 expression by each of the cell lines, Northern blot, nuclear run-on transcription, and immunoblot analyses were used (Fig. 1A–C)Citation . GSTP1 CpG island hypermethylation was detected by Southern blot analysis of genomic DNA after digestion with NotI, a restriction endonuclease that cuts the sequence GCGGCCGC only when it does not contain 5-mCpG (Fig. 1D)Citation . MCF-7 breast cancer cells were found not to express either GSTP1 mRNA or GSTP1 polypeptides, display very low levels of GSTP1 transcription, and contain only hypermethylated GSTP1 CpG island alleles (Fig. 1A–D)Citation . In contrast, MCF-7/ADR breast cancer cells, derived from MCF-7 cells by selection in doxorubicin, expressed abundant GSTP1 mRNA and GSTP1 polypeptides, displayed substantial levels of GSTP1 transcription, and contained only unmethylated GSTP1 CpG island alleles (Fig. 1A–D)Citation . Furthermore, treatment of MCF-7 breast cancer cells with the DNMT inhibitor 5-aza-C, but not treatment with the HDAC inhibitor TSA, stimulated GSTP1 expression within 72 h (Fig. 2)Citation . These data, which suggest that GSTP1 CpG island hypermethylation is responsible for a reduction in GSTP1 transcription in MCF-7 breast cancer cells, support previous observations made by Jhaveri and Morrow (14) .



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  		Fig. 1. GSTP1 CpG island hypermethylation and GSTP1 expression in MCF-7 and MCF-7/ADR breast cancer cells. A, immunoblot analysis for GSTP1 in MCF-7 and MCF-7/ADR cells (10) . Protein extracts from each of the cells were electrophoresed on polyacrylamide gels and then stained with Coomassie blue (Lanes 2 and 3) or transferred to nitrocellulose filters and stained with antibodies to GSTP1 (Lanes 4 and 5). B, Northern blot analysis of GSTP1 and estrogen receptor mRNA (10 , 27) . C, nuclear run-on transcription analysis (8 , 28) . RNA, isolated from cell nuclei that had been incubated with radiolabeled nucleoside triphosphates, was hybridized to filters containing GSTP1, MDR1, and TOPO1 DNA. The amount of radiolabeled RNA hybridizing to GSTP1 and MDR1 DNA, normalized to the amount of RNA hybridizing to TOPO1 DNA, as detected by radioautography, is plotted as relative intensity. D, Southern blot analysis of GSTP1 DNA treated with HindIII, EcoRI, and the 5-mCpG-sensitive endonuclease NotI (10) . The presence of 5-mCpG at the NotI recognition site prevents cutting of a 4.4-kb fragment to a 2.2-kb fragment, detected after hybridization with radiolabeled GSTP1 cDNA.

 


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  		Fig. 2. Restoration of GSTP1 mRNA expression in MCF-7 cells by treatment with 5-aza-C. Northern blot analysis for GSTP1 mRNA was undertaken for MCF-7 cells treated with 10 mM 5-aza-C, 100 ng/ml TSA, or a combination of 5-aza-C and TSA, for 24, 48, and 72 h.

 
To further explore the association of GSTP1 CpG island hypermethylation and GSTP1 expression in MCF-7 breast cancer cells, stable MCF-7 subclones, isolated after prolonged treatment with 5-aza-C, were subjected to Southern and Northern blot analyses (Fig. 3, A and B)Citation . The different MCF-7-aza-C subclones displayed variable reductions in GSTP1 CpG island hypermethylation, as evidenced by variable cutting of GSTP1 transcriptional promoter sequences with NotI (Fig. 3A)Citation and variable increases in GSTP1 mRNA expression (Fig. 3B)Citation . To ascertain whether differences in the patterns of GSTP1 CpG island hypermethylation among the MCF-7-aza-C subclones might explain differences in GSTP1 expression, genomic DNA from HMECs, MCF-7 and MCF-7/ADR breast cancer cells, and MCF-7-aza-C clones 5 and 7 was subjected to bisulfite genomic sequencing, capable of discriminating 5-mCpG from CpG at GSTP1 CpG islands (Refs. 8 , 31 , and 32 ; Fig. 3CCitation ). As expected for normal human cells, the GSTP1 CpG island alleles in HMECs were without significant CpG dinucleotide methylation (34) . For MCF-7 breast cancer cells, devoid of GSTP1 mRNA, the GSTP1 CpG islands were densely methylated. In contrast, for MCF-7/ADR breast cancer cells, which expressed high levels of GSTP1 mRNA, almost no methylated CpG dinucleotides were present at GSTP1 CpG island alleles. When genomic DNA from MCF-7-aza-C clone 5, which expressed low levels of GSTP1 mRNA, and MCF-7-aza-C clone 7, which expressed high levels of GSTP1 mRNA, were analyzed by bisulfite genomic sequencing, a reduction in GSTP1 CpG island methylation, in comparison with MCF-7 cells, was evident for each 5-aza-C-treated MCF-7 subclone. However, MCF-7-aza-C clone 7, which produced GSTP1 mRNA at levels comparable with MCF-7/ADR cells, appeared to contain GSTP1 CpG island alleles that were free of 5-mCpG, like MCF-7/ADR cells, whereas MCF-7-aza-C clone 5, which expressed far less GSTP1 mRNA, contained GSTP1 CpG island alleles with a 5-mCpG density intermediate between MCF-7 and MCF-7/ADR cells. These findings support a role for the density of 5-mCpG dinucleotides at the GSTP1 CpG island in the regulation of GSTP1 transcription. Fully, or nearly fully, methylated GSTP1 CpG islands appeared transcriptionally silent, incompletely methylated GSTP1 CpG islands appeared transcriptionally inhibited, and unmethylated GSTP1 CpG islands appeared capable of high transcription activity.



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  		Fig. 3. GSTP1 CpG island hypermethylation and GSTP1 mRNA expression in 5-aza-C-treated MCF-7 subclones. Independent subclones were isolated by limiting dilution from cultures of MCF-7 cells after treatment for many months with 5-aza-C. A, Southern blot analysis of GSTP1 DNA from MCF-7 cells, MCF-7/ADR cells, and MCF-7-aza-C clones, after treatment with HindIII, EcoRI, and NotI. B, Northern blot analysis of GSTP1 mRNA expression. C, bisulfite genomic sequencing analysis of GSTP1 methylation using genomic DNA from normal HMECs (Clontech), MCF-7 cells, MCF-7/ADR cells, and MCF-7-aza-C clones 7 and 5 (8) . Displayed are 5-mCpG (black dots) and CpG (gray dots) present in GSTP1 alleles.

 
The effect of GSTP1 CpG island hypermethylation on GSTP1 transcriptional promoter activity was also assessed using transient transfection experiments. In preliminary studies, a series of GSTP1 promoter/CAT reporter constructs were transfected into MCF-7 breast cancer cells (Fig. 4A)Citation . Results indicated that an AP-1 recognition sequence at -69 to -63 5' of the transcription start site was required for GSTP1 promoter function and that a transcriptional repressor likely bound between -105 and -85 5' of the transcription start site. These findings are in agreement with data collected by Moffat et al. (23 , 24) . However, when GSTP1 promoter sequences were treated with SssI, a CpG methylase, ligated to luciferase reporter sequences and then transfected into MCF-7 breast cancer cells, a significant reduction in luciferase reporter activity was evident for a construct carrying 5-mCpG dinucleotides at the GSTP1 promoter (Fig. 4B)Citation . Of interest, treatment of transfected MCF-7 cells with TSA appeared to increase luciferase reporter expression >7-fold when the GSTP1 promoter was unmethylated, consistent with some role for HDACs in the regulation of unmethylated GSTP1 promoters (Fig. 4B)Citation . TSA treatment also increased luciferase reporter expression when the GSTP1 promoter was methylated, but not when the AP-1 recognition site was deleted, suggesting that HDAC might contribute in some way to the repression of GSTP1 transcription associated with CpG island hypermethylation (Fig. 4B)Citation . Nonetheless, TSA treatment did not increase the activity of methylated GSTP1 promoters to the level of unmethylated GSTP1 promoters, suggesting that factors other than HDACs might also be involved in the repression of GSTP1 transcription associated with GSTP1 CpG island hypermethylation (Fig. 4B)Citation . Of note, TSA was also not able to elevate GSTP1 expression in MCF-7 cells carrying hypermethylated GSTP1 CpG islands (Fig. 2)Citation . TSA readily augmented the function of an SV40 promoter/luciferase reporter construct (Fig. 4B)Citation .



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  		Fig. 4. Effects of CpG dinucleotide methylation on GSTP1 promoter activity in MCF-7 cells. In A, a series of GSTP1 promoter-CAT reporter constructs were evaluated by transfection into MCF-7 cells as described previously (8 , 24) . CAT activity, relative to pGSTP-1, is shown. In B, GSTP1 promoter sequences ligated to luciferase reporters were treated with the CpG methylase SssI, or left untreated, and then directly transfected into MCF-7 cells without propagation in Escherichia coli (8) . After transfection, the cells were treated with TSA (100 ng/ml) for 48 h or left untreated. Luciferase activity, relative to unmethylated GSTP1 promoter sequences, is plotted. As controls, a truncated GSTP1 promoter/luciferase reporter construct (pGL3_GSTP-6U) and an SV40 promoter/luciferase reporter (pGL3Control) construct were also examined.

 
To determine whether MBD family proteins were bound to hypermethylated GSTP1 CpG islands in MCF-7 breast cancer cells, chromatin from MCF-7 cells was fixed lightly with formalin, liberated by cell lysis, sonicated to reduce average DNA fragment size, and then subjected to immunoprecipitation with antibodies against acetylated histone H4, MBD family proteins MeCP2 and MBD2/3, and DNMT1 (33) . When DNA in the immunoprecipitates was analyzed by quantitative PCR amplification, GSTP1 promoter sequences were recovered along with MBD2/3 and with DNMT1 but not with acetylated H4 or with MeCP2 (Fig. 5B)Citation . In contrast, MDR1 promoter sequences in MCF-7 cells were recovered along with acetylated H4, MeCP2, and MBD2/3 (Fig. 5C)Citation . For MCF-7/ADR breast cancer cells, which contain unmethylated GSTP1 promoter sequences and express high levels of GSTP1 mRNA, ChIP analysis revealed an association of acetylated histone H4 with the GSTP1 promoter, consistent with active GSTP1 transcription, but no association of GSTP1 promoter sequences with MeCP2, MBD2/3, or DNMT1 (Fig. 5B)Citation . These data suggest that MBD2/3, DNMT1, or both might be responsible for GSTP1 repression seen in association with GSTP1 CpG island hypermethylation in MCF-7 breast cancer cells.



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  		Fig. 5. ChIP analysis of the GSTP1 promoter in MCF-7 and MCF-7/ADR cells. A, PCR primers used for detection of GSTP1 and MDR1 promoter DNA in ChIP assays. B, ChIP targeting GSTP1 promoter in MCF-7 and MCF-7/ADR cells using antibodies against acetylated H4 histone, MeCP2, MBD2/3, and DNMT1. The amount of target GSTP1 promoter DNA, estimated by quantitative PCR and recovered by immunoprecipitation, relative to the total amount of GSTP1 promoter DNA subjected to precipitation, is plotted as "Fraction of Total." C, comparative ChIP analysis targeting GSTP1 versus MDR1.

 
In a direct test of whether MBD2 mediated GSTP1 repression in MCF-7 cells, siRNA complementary to MBD2, MBD3, MeCP2, DNMT1, and lamin A/C mRNAs were repeatedly transfected into MCF-7 breast cancer cells, resulting in a ~79% reduction in MBD2 polypeptides, an ~83% reduction in MeCP2 polypeptides, and a ~77% reduction in DNMT1 polypeptides, respectively, within 5 days by immunoblot analysis (Fig. 6ACitation ; Ref. 30 ). When the effect of siRNA treatment on GSTP1 mRNA expression was assessed, using quantitative reverse transcriptase-PCR, an increase in GSTP1 mRNA levels was seen only for MCF-7 cells treated with siRNA targeting MBD2 or DNMT1 mRNA (Fig. 6B)Citation . Of note, the level of GSTP1 mRNA found in MCF-7 cells treated with siRNA targeting either MBD2 or DNMT1 mRNA was substantially less than that found in 5-aza-C-treated MCF-7 cells. Whether this was because MBD2 or DNMT1 polypeptide levels were insufficiently reduced by siRNA treatment, or because other proteins contributed to GSTP1 repression, was not ascertained. Nonetheless, taken together, all of the data collected suggest that MBD2, perhaps along with DNMT1, binds to hypermethylated GSTP1 CpG island sequences in MCF-7 breast cancer cells to prevent GSTP1 mRNA expression.



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  		Fig. 6. siRNA knock-down of 5-mCpG binding proteins and GSTP1 mRNA expression in MCF-7 cells (30) . siRNAs targeting mRNAs encoding MBD2, MBD3, MeCP2, DNMT1, and lamin A were repeatedly transfected into MCF-7 cells. In A, immunoblot analyses of MDB2, MeCP2, and DNMT1 are displayed. B, GSTP1 mRNA expression in MCF-7 cells treated with siRNAs. GSTP1 mRNA levels were estimated using quantitative reverse transcriptase-PCR (16) . Untreated MCF-7 cells served as a negative control; MCF-7 cells treated with 5 µM 5-aza-C for 72 h served as a positive control.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MeCP2 contains a ~70 amino acid minimal region that mediates selective binding to DNA containing 5-mCpG (34 , 35) . Similar MBD motifs are present in MBD1, MBD2, MBD3, and MBD4 (36 , 37) . MeCP2 also contains a transcriptional repression domain that permits interaction with the transcriptional repressor Sin3 and associated HDACs (38, 39, 40) . MeCP2 can thus act as a CpG island methylation-dependent transcriptional repressor by binding transcriptional regulatory sequences carrying 5-mCpG and recruiting HDACs. For this reason, MeCP2-mediated inhibition of 5-mCpG-containing promoter activity has been found to be alleviated by treatment with TSA, an inhibitor of HDACs (39 , 40) . In the current study, MeCP2 did not appear to be responsible for inhibition of transcription associated with GSTP1 CpG island hypermethylation in MCF-7 breast cancer cells, because MeCP2 was not present at hypermethylated CpG island sequences near the transcriptional promoter, and neither TSA treatment nor siRNA-promoted reduction in MeCP2 levels was able to restore GSTP1 mRNA expression.

Instead, MBD2 was detected bound to the hypermethylated GSTP1 promoter in MCF-7 breast cancer cells, and the targeted reduction of MBD2 levels stimulated GSTP1 mRNA expression. MBD2 has been found to be a component of a 1 mega dalton transcription repression complex, MeCP1, which also contains the Mi-2/NuRD chromatin remodeling complex subunits MBD3, HDAC1, and HDAC2; histone-binding proteins RbAp46 and RbAp48; the SWI/SNF helicase/ATPase domain-containing protein Mi2; and MTA2 and in addition contains two uncharacterized polypeptides of Mr 66,000 and 68,000 (41, 42, 43, 44, 45) . MBD3 does not appear to recognize 5-mCpG-containing DNA (36) . As a result, in the absence of MBD2, Mi-2/NuRD complexes, capable of catalyzing ATP-dependent chromatin remodeling, are incapable of selectively binding hypermethylated transcriptional regulatory sequences (42) . In the MeCP1 complex, MBD2 acts to recruit the Mi-2/NuRD chromatin remodeling complex to 5-mCpG-containing DNA (41 , 42) . Consistent with the recruitment of HDAC-containing transcriptional repression complexes to hypermethylated GSTP1 promoters in MCF-7 cells, acetylated histone H4 was detected at with unmethylated GSTP1 promoters in MCF-7/ADR cells but not at hypermethylated GSTP1 promoters in MCF-7 cells. Feng and Zhang (43) reported that the expression of a mutant Mi2, with deficient ATPase activity, in HeLa cells partially prevented repression of transcription from a methylated promoter. Perhaps, MBD2 recruitment of Mi2 nucleosome remodeling activity, present in the MeCP1 complex, along with HDAC activity, may underlie MBD2-mediated repression of hypermethylated GSTP1 promoters in MCF-7 cells.

ChIP detected DNMT1, along with MBD2, selectively bound to hypermethylated GSTP1 promoter sequences in MCF-7 breast cancer cells. In addition, knock-down of DNMT1 polypeptide levels by siRNA treatment triggered GSTP1 mRNA expression. Although the reduction in DNMT1 levels might lead to GSTP1 promoter activity as a result of inadequate maintenance of CpG dinucleotide methylation, Rountree et al. (46) have reported that DNMT1 can establish a transcriptional repression complex via interactions with DMAP1, a transcriptional corepressor that also binds the corepressor TSG101, and HDAC2. Furthermore, DMAP1, which binds the first 121 amino acids in DNMT1, was found to repress transcription in a manner independent of HDACs (46) . We were unable to test whether DNMT1 functioned to repress transcription from hypermethylated GSTP1 promoters in MCF-7 cells, or merely maintained the methylated state of the GSTP1 CpG island, in our experiments. Nonetheless, the recruitment of DNMT1 to GSTP1 promoter sequences in breast cancer cells appeared dependent on CpG dinucleotide methylation, because DNMT1 was not detected in association with unmethylated GSTP1 promoters in MCF-7/ADR breast cancer cells. In late S phase of the replicative cell cycle, DNMT1 has been recovered in a complex with MBD2 and MBD3 that can bind fully methylated and hemimethylated DNA (47) . If DNMT1/MBD2/MBD3 complexes exist both in non-S phase cells and in S phase cells, then DNMT1 might function both to repress transcription from hypermethylated GSTP1 promoters and maintain GSTP1 CpG island hypermethylation during DNA replication. Recently, Di Croce et al. (48) reported that stable complexes, containing DNMTs, were present at promoters targeted for repression by the leukemia-promoting promgelocytic leukemic protein (PML)-retinoic acid receptor fusion protein and that the formation of these complexes was associated with de novo promoter methylation. Whether the presence of DNMT1 at the GSTP1 promoter in MCF-7 breast cancer cells reflects previous participation of DNMT1 in de novo GSTP1 CpG island hypermethylation has not been tested.

All of the data presented here strongly suggest that MBD2, and perhaps DNMT1, participate in the inhibition of transcription from abnormally hypermethylated GSTP1 promoters in breast cancer cells. Most therapeutic strategies aimed at reactivating "silenced" genes in human cancer cells considered thus far have targeted DNMTs or HDACs. However, the clinical use of nucleoside DNMT inhibitors, such as 5-aza-C and 5-aza-dC, has been complicated by myelotoxicity and other side effects (49) . As the mechanisms by which MBD2 acts to prevent transcription of GSTP1, and of other genes inactivated by CpG hypermethylation in cancers, become more fully elucidated, perhaps new cancer treatment or prevention strategies might target the MBD2 transcriptional repression pathway. In support of this notion, Mbd2-/- mice predictably fail to completely repress transcription from exogenously methylated promoters in transient transfection assays (50) . However, other than a maternal behavior defect, the Mbd2-/- mice appear fairly unremarkable, with normal gene imprinting, maintained repression of endogenous retroviral sequences, and no striking ectopic gene expression (50) . MBD2 pathway-targeted treatments might then be able to reactivate "silenced" genes in cancer cells or precancerous lesions with few worrisome side effects. Clearly, more studies will be required to test this prediction.


   ACKNOWLEDGMENTS
 
W.G.N. has a patent (United States patent 5,552,277) entitled "Genetic Diagnosis of Prostate Cancer."


   FOOTNOTES
 
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.

1 Supported by NIH/National Cancer Institute Grant CA 70196. Back

2 To whom reprint requests should be addressed, at The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Room 151, Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231. Phone: (410) 614-1661; Fax: (410) 502-9817; E-mail: bnelson{at}jhmi.edu Back

3 The abbreviations used are: MBD, methyl-CpG-binding domain; DNMT, DNA methyltransferase; HDAC, histone deacetylase; 5-aza-C, 5-aza-cytidine; siRNA, small interference RNA; ChIP, chromatin immunoprecipitation; HMEC, human mammary epithelial cell; TSA, trichostatin A; CAT, chloramphenicol acetyltransferase. Back

Received 4/ 8/02. Accepted 11/13/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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