Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer

Article1 December 2003free access Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer Esteban Ballestar Esteban Ballestar Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Maria F. Paz Maria F. Paz Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Laura Valle Laura Valle Cytogenetics Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Susan Wei Susan Wei Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MI, 65203 USA Search for more papers by this author Mario F. Fraga Mario F. Fraga Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Jesus Espada Jesus Espada Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Juan Cruz Cigudosa Juan Cruz Cigudosa Cytogenetics Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Tim Hui-Ming Huang Tim Hui-Ming Huang Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MI, 65203 USA Search for more papers by this author Manel Esteller Corresponding Author Manel Esteller Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Esteban Ballestar Esteban Ballestar Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Maria F. Paz Maria F. Paz Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Laura Valle Laura Valle Cytogenetics Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Susan Wei Susan Wei Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MI, 65203 USA Search for more papers by this author Mario F. Fraga Mario F. Fraga Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Jesus Espada Jesus Espada Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Juan Cruz Cigudosa Juan Cruz Cigudosa Cytogenetics Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Tim Hui-Ming Huang Tim Hui-Ming Huang Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MI, 65203 USA Search for more papers by this author Manel Esteller Corresponding Author Manel Esteller Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain Search for more papers by this author Author Information Esteban Ballestar1, Maria F. Paz1, Laura Valle2, Susan Wei3, Mario F. Fraga1, Jesus Espada1, Juan Cruz Cigudosa2, Tim Hui-Ming Huang3 and Manel Esteller 1 1Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain 2Cytogenetics Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain 3Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MI, 65203 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6335-6345https://doi.org/10.1093/emboj/cdg604 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Methyl-CpG binding proteins (MBDs) mediate histone deacetylase-dependent transcriptional silencing at methylated CpG islands. Using chromatin immunoprecitation (ChIP) we have found that gene-specific profiles of MBDs exist for hypermethylated promoters of breast cancer cells, whilst a common pattern of histone modifications is shared. This unique distribution of MBDs is also characterized in chromosomes by comparative genomic hybridization of immunoprecipitated DNA and immunolocalization. Most importantly, we demonstrate that MBD association to methylated DNA serves to identify novel targets of epigenetic inactivation in human cancer. We combined the ChIP assay of MBDs with a CpG island microarray (ChIP on chip). The scenario revealed shows that, while many genes are regulated by multiple MBDs, others are associated with a single MBD. These target genes displayed methylation- associated transcriptional silencing in breast cancer cells and primary tumours. The candidates include the homeobox gene PAX6, the prolactin hormone receptor, and dipeptidylpeptidase IV among others. Our results support an essential role for MBDs in gene silencing and, when combined with genomic strategies, their potential to ‘catch’ new hypermethylated genes in cancer. Introduction DNA methylation, the major epigenetic modification of mammalian genomes, plays an active role in transcriptional repression (Cedar, 1988). Over the past 10 years, increasing evidence has emerged surrounding the active role of CpG island hypermethylation of tumour suppressor genes in cancer development and progression (Esteller, 2002). One of the necessary steps in the epigenetic pathway to cancer involves methyl-CpG binding proteins (MBDs) that mediate transcriptional silencing of the hypermethylated gene promoters. The mammalian family of MBD proteins is composed of five members, namely MeCP2, MBD1, MBD2, MBD3 and MBD4. With the exception of MBD4, which is involved in DNA repair, all MBD proteins associate with histone deacetylases (HDACs) and couple DNA methylation with transcriptional silencing through the modification of chromatin (Ballestar and Wolffe, 2001; Wade, 2001; Prokhortchouk and Hendrich, 2002). Moreover, recent research has demonstrated the presence of MBD proteins in mediating the transcriptionally silenced state of several promoters of hypermethylated tumour suppressor genes in cancer, and in imprinted and X-chromosome inactivated genes in normal cells (Magdinier and Wolffe, 2001; Nguyen et al., 2001; Bakker et al., 2002; El-Osta et al., 2002; Fournier et al., 2002). An essential issue concerns the physiological relevance of the existence of four different MBD-containing co-repressor complexes. The most straightforward explanation is that each complex is targeted to a different subset of genes. To understand the specific roles of different MBDs it is of inherent interest to biochemically characterize the MBD-containing complexes. Thus far, MBD3 is the best-characterized member of the MBD family. It has been reported to be an integral component of the Mi-2/NuRD complex (Wade et al., 1999; Zhang et al., 1999) that also contains a nucleosome remodelling ATPase, HDAC1 and HDAC2 and other proteins. Mammalian MBD3, in contrast to its Xenopus homologue, does not selectively bind methylated DNA and in mammals the Mi-2/NuRD complex is targeted to methylated DNA through association with any of the two forms of MBD2: MBD2a or MBD2b. This combination of Mi-2/NuRD and MBD2 may be synonymous of the MeCP1 complex, the first identified to display binding activity towards methylated DNA (Feng and Zhang, 2001). The Mi-2/NuRD complex may also interact with other sequence-specific DNA binding proteins to cause transcriptional repression. In the case of MBD1, although early reports suggested HDAC-dependent repression (Ng et al., 2000), recent data indicate that MBD1 represses transcription in an HDAC-independent manner that involves association with a novel chromatin-associated factor (Fujita et al., 2003). An alternative approach in understanding the biology of MBD proteins arises from the study of MBD targets. Reports on MBD association to methylated loci have revealed a complex picture. On the one hand, a single association of MBD proteins to methylated loci (Magdinier and Wolffe, 2001; Bakker et al., 2002) has been described. However, multiple recruitment of MBD proteins has been reported in several genes (Fournier et al., 2002; Koizume et al., 2002). Early studies indicated significant differences in the association of MBDs to DNA. For instance, MBD2/MeCP1 is released from nuclei by low salt, suggesting that it is not stably complexed with DNA and seems to require densely methylated DNA. That aside, MBD1 can also affect transcription from unmethylated and hypomethylated promoters (Fujita et al., 2000). The reasons behind the specific role of each MBD-containing complex remain to be explored. The use of chromatin immunoprecipitation (ChIP) with antibodies specific for a particular MBD together with immunocytological analysis potentially provide powerful tools to identify the set of genes controlled by each particular MBD-containing complex and therefore a way to understand the individual roles of each of these complexes. As a first step towards determining the targeting of MBD proteins in a genome-wide context, we have combined individualized studies for each MBD (i.e. ChIP assays for several promoters of tumour suppressor genes) with global genomic approaches [5-methylcytosine (mC) analysis of the MBD-immunoprecipitated products and comparative genomic hybridization]. Most importantly, we have also combined ChIP analysis with a CpG island microarray to define the global profile of MBD targeting. Our studies show the existence of a unique pattern of MBD-binding sites in transformed cells and unmask a new set of epigenetically silenced genes in cancer. Results A gene-specific profile of MBDs exists in hypermethylated CpG island promoters To investigate the involvement of MBDs in epigenetic repression, we initially adopted a candidate gene approach to study genes known to be hypermethylated in a breast tumour model, specifically in the breast cancer cell lines MCF7 and MDA-MB-231. ChIP analyzes were performed using antibodies raised against epitopes unique to each MBD protein (MeCP2, MBD1, MBD2 and MBD3). We have recently validated these antibodies in investigating the recruitment of MBD proteins to imprinted-methylated loci (Fournier et al., 2002). We first performed western blot analysis with these antibodies in all the cell lines studied to ensure that the MBD proteins were indeed expressed (Figure 1A). ChIP assays were then performed in order to identify which particular MBD protein was associated with methylated DNA sequences in MCF7 and MDA-MB-231 cells. Additionally, normal lymphocytes, representing non-tumoural non-cultured cells, and non-transformed lymphoblastoid cell lines were also used as negative controls. Figure 1.ChIP analysis of the occupancy by MBD proteins and histone modification status of several hypermethylated promoters. (A) MBD antisera αMeCP2 N-t, αMBD1 C-t, αMBD2 N-t, αMBD3 C-t were tested on MCF7 and MDA-MB-231 nuclear extracts. On the left, molecular size bands of a pre-stained standard (Kaleidoscope, BioRad) are indicated. Comparable results were obtained with MDA-MB-231 nuclear extracts. (B) A summary of the methylation status of the studied promoters in MCF7 and MDA-MB-231 (extracted from Esteller, 2002). (C) MBD occupancy analysed by ChIP assay. Input and ‘unbound’ fraction of the no antibody (NAB) control are shown followed by the four ‘bound’ fractions for each antibody. ChIP assays shown correspond to MCF7 and MDA-MB-231 cells and isolated lymphocytes. Results with lymphoblastoid cell lines are undistinguishable from those obtained with control lymphocytes. Three groups of sequences are shown: CpG islands tumour suppressor genes, an imprinted gene (IGF2) and repetitive sequences (Sat2 and NBL2). (D) Analysis of the acetylation status of each promoter. Commercial anti-acetylH3 and anti-acetylH4 were used (Upstate Biotechnologies). Control cells and 5-azadC treated cells are shown. (E) Analysis of the methylation status of K9 of H3 is studied. An H3 antibody to a branched peptide with four fingers of the K9-dimethylated TARKST sequence (Lachner et al., 2001) was used. Download figure Download PowerPoint Three types of DNA sequences were analyzed: the CpG islands in the promoter of six tumour suppressor genes, for which DNA methylation and expression status is well characterized (Esteller et al., 2001; Paz et al., 2003a), the CpG island of the imprinted gene IGF2 (insulin-like growth factor 2) and two different repetitive sequences, namely Sat2 (satellite 2) and NBL2 (a non-satellite repeat). The tumour suppressor genes explored were the Ras association domain family 1A gene (RASSF1A), the glutathione S-transferase P1 (GSTP1) gene, the retinoic acid receptor B2 gene (RARB2), the breast cancer 1 gene (BRCA1), the O6-methylguanine-DNA methyltransferase (MGMT) gene and the mutL-homologue 1 (MLH1). A summary of the methylation status of the promoters of these genes is shown in Figure 1B. MBD binding was not observed in any unmethylated promoter. However, a specific profile of MBD occupancy was found for the methylated CpG islands. Whilst MeCP2 and MBD2 were both present in the methylated GSTP1 promoter, for RASSF1A and RARB2 only MeCP2 was bound, and for BRCA1 and MGMT the only MBD present was MBD2 (Figure 1C). In none of the six selected promoters did we find any association with MBD1 or MBD3. In contrast, these six promoters are unmethylated in lymphocytes and lymphoblastoid cell lines and do not exhibit any binding of MBDs in these normal cells (Figure 1C). As a positive control, MBD association to the methylated regions of the IGF2 imprinted gene and the repetitive sequences Sat2 and NBL2 was observed for all the cells analyzed (MDA-MB-231, MCF7, normal lymphocytes and lymphoblastoid cell lines) (Figure 1C). The presence of DNA methylation and MBD association was accompanied by histone deacetylation and lysine 9 histone H3 methylation at these promoters (Figure 1D and E). The restoration of histone acetylation and demethylation on histone H3 both occurred rapidly with the use of the demethylating agent 5-aza-2-deoxycytidine. Consistent with our results and previous findings (Nguyen et al., 2002), we also found that the promoters that were unmethylated and devoid of MBDs were enriched in acetylated histones and H3 lysine 9 was demethylated (Figure 1D and E). MBDs immunoprecipitate methylated DNA The candidate gene approach to identify MBD targets, although necessary, is time consuming. Therefore, to overcome this drawback in a global screen for MBD targets, we employed three independent genomic approaches, using as starting material the immunoprecipitated DNA for each MBD. We first analyzed, by high performance capillary electrophoresis (Fraga and Esteller 2002), the total mC DNA content of each MBD-immunoprecipitated DNA in MCF7 cells. An average 3- to 5-fold enrichment in mC DNA in this MBD–ChIP DNA versus the input DNA was observed (Figure 2), strongly supporting the notion that MBD proteins are tightly associated in vivo with methylated DNA sequences. Furthermore, a gradient of mC DNA content versus the overall amount of bound DNA was observed, with MeCP2 being the protein that binds to the highest ratio of methylated cytosines in the human genome, and MBD3 the lowest (Figure 2). These results further support the differential distribution of MBD binding within the genome. Figure 2.Analysis of global DNA methylation by HPCE of MBD-immunoprecipitated samples. Two electropherograms, corresponding to the input fraction, the MBD1 and MBD2 immunoprecipitated DNAs from MCF7 cells, are shown. The graph shows the methylcytosine content in the same fractions. Download figure Download PowerPoint MBDs are associated with extensive chromosomal regions We further explored the distribution of each MBD protein throughout particular chromosomal regions by using a modification of the comparative genomic hybridization (CGH) protocol (Cigudosa et al., 1998). We labelled the DNA from MCF7 and MDA-MB-231 immunoprecipitated with each one of the MBDs antibodies, and then was competitively hybridized against input DNA from these cells (Figure 3A). We found that the MBD-immunoprecipitated sequences, seen as thick green segments in the CGH karyotype, were unevenly distributed along the chromosomes, yielding a characteristic pattern with broad megabase-length bands (Figure 3B). It is noteworthy that this pattern of banding differs from the one obtained when MDA-MB-231 or MCF7 DNA is competitively hybridized against DNA extracted from normal cells (see, for example, Xie et al., 2002), discarding artifacts due to the aneuploidy of these cancer cells. A remarkable finding is that many of the resulting bands are shared between both the different MBDs and also the two cell lines studied (Figure 3B, see inset). For instance, there were common specific regions of chromosomes 1 (at band 1q13), 9 (9p13), 11 (11q13), 13 (13q21), 15 (15q13), 16 (16p11.2), 17 (17q11.2), 19p, 21 (21q11.2), and 22q recurrently enriched in ChIP isolated sequences in both cell lines (Figure 3B, see inset). Quantitative differences in MBD distribution, however, were still very apparent as the number of regions that contained immunoprecipitated sequences was higher for MBD3 and MeCP2 (53 and 48 segments) versus those obtained with MBD1 and MBD2 (34 and 37 segments). Furthermore, a quantitative difference was also observed between both breast cancer cell lines: chromosomes in MDA-MB-231 contained a much higher proportion of MBD–ChIP sequences compared with MCF7, reflecting the higher number of hypermethylated CpG islands present in MDA-MB-231 versus MCF7 (Paz et al., 2003a). Our CGH data suggest that MBDs tend to be clustered at certain chromosomal loci, an observation that is consistent with the appearance of nuclear domains obtained with MBD antisera in immunolocalization experiments (Hendrich and Bird, 1998). We have observed that immunostaining with anti-MBD antisera produces a combined pattern of discrete foci in a context of diffuse nuclear staining (data not shown). The diffuse MBD pattern colocalizes with mC staining (especially in the case of MBD1) and some of the MBD foci are also coincident with mC spots (data not shown). This observation may reflect that some of the observed MBD nuclear distribution is due more to architectural features of the nucleus than to genome sequence patterns. Figure 3.Comparative genomic hybridization of MBD-immunoprecipitated DNAs in metaphase chromosomes. (A) Diagram showing the combination of comparative genome hybridization with the ChIP assay. (B) Ideograms showing hybridization of ChIP DNAs on the human karyotype. Thick vertical lines on either side of the chromosome ideogram indicate only recurrent MBD enrichment (green) or exclusion (red) of a chromosome or a chromosomal region. The four green and four red lines correspond, respectively, to MBD1, MBD2, MBD3 and MeCP2 from inside to outside. The analysis shown corresponds to MDA-MB-231 samples. The inset shows three chromosomes from the MCF7 samples. Download figure Download PowerPoint MBDs identify novel hypermethylated genes in cancer The resolution of CGH and immunolocalization does not allow the identification of specific sequences associated with MBD proteins. To obtain an accurate and comprehensive profile of the genes targeted by MBDs in human breast cancer, we have combined chromatin immunoprecipitation and array technology (ChIP on chip). We have probed the MCF7 and MDA-MB-231 DNA from ChIP assays using the four different MBD antisera described above with a DNA microarray that contains a library of 7777 CpG islands. This CpG island microarray provides a high throughput method for the identification of in vivo nuclear factor targets, as has been demonstrated for E2F (Weinmann et al., 2002). Three independent hybridizations of the CpG island microarray with three independent chromatin-immunoprecipitated samples were performed for each MBD protein in each of the cell lines. From a purely quantitative standpoint, MBD2 produced the highest number of positive clones, supporting the in vitro data that showed that MBD2 has the highest affinity for methylated DNA 8 (Fraga et al., 2003), followed by MBD1, MBD3 and MeCP2 (Figure 4B). The array hybridization results across all spotted CpG islands show that 7.1% of the clones were positive for all four MBDs, while 24.2, 10.3, 9 and 5.8%, were positive singly for MBD2, MBD3, MeCP2 and MBD1, respectively (Figure 4C). Therefore, the data indicate that MBD target sequences tend to be occupied by either a single MBD (preferentially MBD2) or all of them, rather than double or triple occupancy. Figure 4.(A) Representative microarray images for different MBD-immunoprecipitated MDA-MB-231 samples. Cy5 labelled chromatin DNAs were hybridized to CpG island microarray as described in the text. The hybridization images were acquired and normalized signal intensities of hybridized spots were compared to those of the control (total input). (B) Graph showing the percentage of total positive clones (relative to the total input control) obtained for each MBD-immunoprecipitated DNA. (C) Graph showing the percentage of positive clones for the different combinations: all MBD proteins, three MBDs, two MBDs or a single MBD. (D) Confirmation of MBD targets by individual ChIP analysis and PCR with primers designed to the individual loci. Input, no antibody (unbound fraction) and MBD immunoprecipitated fractions (bound) are shown. Download figure Download PowerPoint In order to identify the genes immunoprecipitated by the MBDs, we sequenced 60 CpG island-positive clones that were common to three independent experiments: 10 from the subgroup immunoprecipitated by all MBDs, 10 from each of the four subgroups immunoprecipitated by a unique MBD, and 10 from a subgroup immunoprecipitated by both MBD2 and MBD3. This last subset was chosen upon the basis of reports regarding MBD2 and MBD3 links within the MeCP1 complex (Feng et al., 2001; Hendrich et al., 2001). Table I summarizes the sequencing data and blast search results. The list of novel MBD target genes in MCF7 and MDA-MB-231 breast cancer cells includes excellent candidate genes for the malignant phenotype. Some of these genes have already been associated with cancer, such as the prolactin hormone receptor (PRLR) (Jonathan et al., 2002), the protein- tyrosine phosphatase non-receptor type (PTPN4) (Ogata et al., 1999), the dipeptidyl peptidase IV (DPPIV) (Pethiyagoda et al., 2000) and PIP5K (Chatah and Abrams, 2001). It was essential to confirm that the identified clones were bound by MBDs in vivo and the results from individual ChIPs with MCF7 and MDA-MB-231 cells with the four MBD antibodies confirmed the results from the CpG island microarray (Figure 4D). For sequences immunoprecipitated with MeCP2 antiserum, results were confirmed using the corresponding commercial antibody (data not shown). As a negative control, we performed individual ChIP analysis with isolated lymphocytes and lymphoblastoid cell lines and we found that MBDs were absent in the promoters of these genes. Table 1. MBD targets identified by ChIP-CpG microarray analysis Gene Accession No. Location MBD Present in other cell line MCF7 cells clone RP11–403I13 AL356957 MeCP2 Also MDA-MB-231 clone RP11–99O17 AC018706 7p15.2 MeCP2 DKFZp686D16148 AL701693 19q13.42 MeCP2 Also MDA-MB-231 enpp4 AL035701 6p21.1 MeCP2 FLJ00132 AK074061 11q12.2 MeCP2 shoygo (ax09h10) BG941206 4p14 MBD1 Also MDA-MB-231 bing4 NM_005452 6p21.32 MBD1 clone RP11–576P10 7p11.2-p21 MBD1 pak2 3p24.3 MBD2 clone RP1-77N19 Z98886 1p36.2–36.3 MBD2 tbx19 AJ010277 1q23-q24 MBD2 atp5i BAA78778 4p16.3 MBD2 Also MDA-MB-231 cox6c P09669 8q22-q23 MBD3 Also MDA-MB-231 LOC199704 XM_113994 19q13.13 MBD3 leng6 AF211971 19q13.4 MBD3 Also MDA-MB-231 clone CTC-454I21 AC012309 19q13.13 MBD3 stearoyl-CoA desaturase BAA93510 10q24.33 MBD3 Paired box protein pax-6 CAB05885 11p13 MBD3 Also MDA-MB-231 mps-1 A48045 MBD2 + MBD3 Also MDA-MB-231 cox6c P09669 8q22-q23 MBD2 + MBD3 ptpn4 NM 002830 2q14.1 MBD2 + MBD3 Also MDA-MB-231 efna5 P52803 5q21 All MBDs bcat2 NM_001190 19q13 All MBDs dppiv P27487 2q24.3 All MBDs MDA-MB-231 cells clone: IMAGE:4550691 BG331429 4p16.3 MeCP2 pip5k BC007833 1q22-q24 MeCP2 FLJ32618 AK057180 16p13.13 MeCP2 FLJ32618 AK057180 16p13.13 MeCP2 FLJ32760 AK057322 2q37.3 MeCP2 CS0DJ015YH07 AL558734 9p13.3 MeCP2 oatp-d AB031050 15q26.1 MBD1 clone YB67A06 AF147380 1q31.1 MBD1 ptprm NM_002845 18p11.23 MBD1 clone RP1-207F6 AL133258 6p22.1 MBD2 kcnk10 NM_021161 14q31.3 MBD2 prlr NM_000949 5p13.3 MBD2 clone RP5-947L8 AL355178 1p34.1–36.11 MBD2 clone RP13–202B6 AL591625 Xp21.1–21.3 MBD3 bat5 NM_021160 6p21.33 MBD3 DKFZp564E1878 (hspc228) AL136684 12q13.3 MBD2 + MBD3 HSPC228 NM_016485 6q24.1 MBD2 + MBD3 Also MCF7 HSPC228 NM_016485 6q24.1 MBD2 + MBD3 Also MCF7 mef2a NM_005587 15q26.3 MBD2 + MBD3 Also MCF7 alox12b AF038461 17p13.1 All MBDs Apolipoprotein E precursor (Apo-E) P02649 16p11.2 All MBDs Also MCF7 clone: IMAGE:3086912 BF509821 11q12.3 All MBDs Beta crystallin B2 (crybb2) P43320 22q11.23 All MBDs FLJ14146 NM_024709 1q42.12 All MBDs Once their association to MBD proteins was confirmed, this subset of genes was subjected to further characterization for DNA methylation and expression. Bisulfite genomic sequencing spanning their corresponding CpG islands was used to demonstrate hypermethylation in the corresponding breast cancer cell lines where MBD binding had been confirmed by ChIP analysis (MCF7, MDA-MB-231 or both) (Figure 5A and C). Furthermore, these results were confirmed by methylation-specific PCR analysis (Figure 5C). DNA extracted from isolated lymphocytes and lymphoblastoid cell lines was also analyzed for the methylation status of all the above genes by both bisulfite genomic sequencing and methylation-specific PCR. In both cases, all these genes were unmethylated in these normal cells in agreement with the absence of MBD binding previously demonstrated. Figure 5.DNA methylation and expression analysis of the particular genes found using the ChIP on chip approach. (A) Bisulfite genomic sequencing of the PRLR CpG island demonstrating hypermethylation in MDA-MB-231 cells and hypomethylation in MCF7 cells. On the contrary, the ENPP4 CpG island is hypermethylated in MCF7 cells but not in MDA-MB-231 cells. A fragment of the sequence is shown. Unmethylated Cs become Ts upon bisulfite modification. Below, a schematic representation of some of the CpG sites included in the PCR fragment is shown. CpG sites are represented as circles that are black when methylated. (B) Methylation-specific PCR confirms the presence of hypermethylation in ENPP4 and PRLR primary breast tumours. A number of cases are shown, indicated as c1–c9. (C) Summary table with the bisulfite sequence, methylation-specific PCR results for MCF7, MDA-MB-231 and primary breast tumours. Dark and light shading indicates methylation or no methylation, respectively. (D) Expression analysis monitored by RT–PCR after co-amplification with GAPDH. In all panels, the top band (400 bp) is GAPDH and the bottom band (of ∼200 bp) is each of the specific genes. Download figure Download PowerPoint To address the functional consequences of the CpG island promoter hypermethylation in association with the binding of MBDs, we performed expression analysis of the described genes. In all analyzed cases, we found that genes with unmethylated sequences and no MBD binding were expressed, while hypermethylated MBD-associated genes were transcriptionally silenced. In these latter cases, the use of 5-aza-2′-deoxycytidine was able to restore gene expression (Figure 5D). Furthermore, we demonstrated that this CpG island hypermethylation was not merely a feature of these particular breast cancer cell lines, but was present in a significant proportion of human primary breast carcinomas (Figure 5B and C). Two MBD-associated genes, PAX6 and PRLR, inhibit growth in MDA-MB-231 cells To gain further insight into the potential role of some hypermethylated genes identified by our ChIP on chip strategy of genomic screening, we decided to reintroduce some of these genes into the MDA-MB-231 cell line, as carried out for other hypermethylated tumour suppressor genes (Tomizawa et al., 2001; Yoshikawa et al., 2001). Expression vectors containing PAX6 and PRLR were independently used to transfect MDA-MB-231 cells, in which both genes were silenced in association with methylation. As positive control for growth inhibition, we used wild-type p53. A mutant form of p53 (N175H) was used as an additional negative control, besides empty vector. Forty-eight hours after