Oncogene (2008) 27, 3556–3566 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of methyl-CpG-binding domain proteins L Lopez-Serra1, E Ballestar1, S Ropero1, F Setien1, L-M Billard2, MF Fraga1, P Lopez-Nieva1, M Alaminos3,4, DGuerrero 5, R Dante2 and M Esteller1 1Cancer Epigenetics Laboratory, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 2Unite´ INSERM 590, Laboratoire d’Oncologie Mole´culaire, Centre Le´on Be´rard, Lyon, France; 3Department of Histology, Granada University, Granada, Spain; 4Fundacio´n Hospital Clı´nico, Granada, Spain and 5Centro de Investigacio´n Biome´dica, Servicio Navarro de Salud, Navarra, Spain Methyl-cytosine-phosphate-guanine (CpG)-binding do- ‘read’ DNA methylation patterns may play a central main (MBD) proteins are bound to hypermethylated role in cellular transformation. This is mainly due to the promoter CpG islands of tumor suppressor genes in recognition that DNA hypermethylation of the promo- human cancer cells, although a direct causal relationship ter cytosine-phosphate-guanine (CpG) islands of tumor at the genome-wide level between MBD presence and gene suppressor genes, and resulting gene inactivation, silencing remains to be demonstrated. To this end, we have represent a major event in tumor etiology and progres- inhibited the expression of MBD proteins in HeLa cells sion (Jones and Laird, 1999; Herman and Baylin, by short hairpin RNAs; and studied the functional 2003; Esteller, 2007). The methyl-CpG-binding domain consequences of MBD depletion using microarray-based (MBD) family of proteins is the largest group of these expression analysis in conjunction withextensive bisulfite factors that bind methylated DNA, and their exact role genomic sequencing and chromatin immunoprecipitation. in the epigenetic silencing of tumor suppressor genes has The removal of MBDs results in a release of gene functional and translational consequences that need to silencing associated witha loss of MBD occupancy in be clarified (Ballestar and Esteller, 2005; Fatemi 50-CpG islands without any change in the DNA methyla- and Wade, 2006). The MBDfamily of proteins is tion pattern. Our results unveil new targets for epigenetic composed of five bona fide members, MeCP2, MBD1, inactivation mediated by MBDs in transformed cells, such MBD2, MBD3 and MBD4, which share an MBD as the cell adhesion protein c-parvin and the fibroblast that allows them to bind methylated DNA (Hendrich growth factor 19, where we also demonstrate their and Bird, 1998; Ballestar and Esteller, 2005; Fatemi bona fide tumor suppressor features. Our data support and Wade, 2006). With the exception of MBD4, a fundamental role for MBD proteins in the direct which is involved in DNA repair (Hendrich et al., maintenance of transcriptional repression of tumor 1999), and MBD3, whose MBD is unable to bind suppressors and identify new candidate genes for epige- methylated DNA selectively (Saito and Ishikawa, 2002; netic disruption in cancer cells. Fraga et al., 2003), MBDs couple DNA methylation Oncogene (2008) 27, 3556–3566; doi:10.1038/sj.onc.1211022; with transcriptional repression through association with published online 28 January 2008 histone deacetylases (HDACs; Jones et al., 1998; Nan et al., 1998; Wade et al., 1999) and histone methyl- Keywords: DNA methylation; epigenetics; methyl-CpG- transferases (Fujita et al., 2003; Fuks et al., 2003). These binding domain proteins; tumor suppressor genes; RNA properties have led them to being proposed as having a interference major role in the aberrant epigenetic silencing of tumor suppressor genes (Ballestar and Esteller, 2005; Fatemi and Wade, 2006). Genetic analysis of MBDproteins has shown that most single MBD-deficient mouse models do not exhibit Introduction dramatic phenotypes (Guy et al., 2001; Hendrich et al., 2001; Zhao et al., 2003). However, detailed analysis During the past few years, considerable attention has shows that subtle but important changes are associated been focused upon the possibility that proteins that with deficiency in individual MBDproteins. For instance, loss of MBD2 is associated with a significant Correspondence: Dr M Esteller, Cancer Epigenetics Laboratory, change in the abundance of transcripts for certain Molecular Pathology Program, Spanish National Cancer Research cytokines that are crucial to the process of T-lympho- Centre (CNIO), C/Melchor Fernandez Almagro 3, Madrid 28029, cyte differentiation (Hutchins et al., 2002). Crossing Spain. Min E-mail: [email protected] Mbd2-null mice (Hendrich et al., 2001) with Apc Received 12 October 2007; revised 26 November 2007; accepted 4 also inhibits the development of intestinal adenomas December 2007; published online 28 January 2008 (Sansom et al., 2003) and MeCP2 is required for MBD depletion in transformed cells L Lopez-Serra et al 3557 prostate cancer cell growth (Bernard et al., 2006). In the critical role of MBDs in the maintenance of addition, disruption of the MeCP2 gene in Rett epigenetic gene silencing and the usefulness of MBD- syndrome samples from mouse models (Jordan et al., depletion strategies to ‘catch’ new hypermethylated 2007) or human patients (Ballestar et al., 2005) is genes in human cancer. associated with upregulation of a subset of genes as a result of the loss of interaction of MeCP2 with methylated CpG sites at the promoter region. Results Since DNA methylation patterns differ dramatically between cancer cells and their normal counterparts Removal of MBD proteins results in release of epigenetic (Jones and Laird, 1999; Herman and Baylin, 2003; gene silencing Esteller, 2007), it is likely that the distribution and, To demonstrate functionally the direct role of MBDs in ultimately, the biological relevance of MBDproteins are gene silencing, we carried out a systematic depletion of also significantly different in normal and transformed MBDproteins in HeLa cells (human cervical cancer) in cells. In normal cells, promoter CpG islands are mostly conjunction with comprehensive expression microarray unmethylated, with the exception of those of imprinted analyses. We interfered with the expression of the three genes, X-chromosome genes in females and a number of MBD proteins (MeCP2, MBD1 and MBD2) that have a tissue-specific genes (Jones and Laird, 1999; Herman functional MBDand for which association with histone and Baylin, 2003; Esteller, 2007). In contrast, cancer modification enzymes and transcriptional repression cells are characterized by the generation of specific properties has been demonstrated (Figure 1a; Jones patterns of hypermethylation at the promoter CpG et al., 1998; Nan et al., 1998; Ng et al., 1999, 2000). islands of tumor suppressor genes (Jones and Laird, Seven different RNA interference experiments were 1999; Herman and Baylin, 2003; Esteller, 2007). DNA- performed: single MeCP2, MBD1 and MBD2, com- methylated heterochromatic sequences are probably the bined MeCP2/MBD1, MeCP2/MBD2 and MBD1/ primary binding site for MBDproteins in normal cells, MBD2, and the triple MeCP2/MBD1/MBD2 combina- as suggested by the fact that MeCP2 is enriched in tion. First, we confirmed the robust depletion of the pericentromeric heterochromatin in murine cells, in corresponding MBDproteins after transient transfec- accordance with its content of major satellite DNA, tion with RNAi oligonucleotides at both the RNA the largest fraction of methylated DNA sequences in (Figures 1b and c) and protein levels (Figure 1d) mice (Lewis et al., 1992). Another normal set of targets for the single, double and triple combinations. Upon for MBDs are imprinted genes, where MBDs associate depletion of one single MBD, we did not observe any with the differentially methylated allele (Fournier et al., significant changes in the expression of the other 2002). In cancer cells, the hypermethylated promoter members of the MBDfamily (Figures 1b and d). No CpG islands of tumor suppressor genes constitute new significant changes in the global 5-methylcytosine DNA and aberrant targets for MBDproteins and accumu- content determined by high-pressure liquid chromato- lated evidence indicates that hypermethylation of tumor graphy were observed upon MBDdepletion (Supple- suppressor genes is accompanied by association of mentary Figure S1). From the HeLa-untreated and MBDs (Magdinier and Wolffe, 2001; Nguyen et al., MBD-interfered cells, total RNA was extracted, reverse 2001; Bakker et al., 2002; Koizume et al., 2002; Ballestar transcribed, hybridized to cDNA microarrays and the et al., 2003; Lopez-Serra et al., 2006). Given the data were analysed, as described in ‘Materials and association of MBDs with HDACs and methyltrans- methods’. ferases and their DNA methylation-dependent effects on We observed a release of transcriptional silencing gene transcription, their essential contribution to the upon depletion of MBDproteins by RNAi (Figure 2a). epigenetic silencing of tumor suppressor genes is Of the 6386 genes represented in the expression array, generally accepted, even though no formal evidence 967 (15%) experienced an expression change between has been presented to date. untreated and triple MBD-depleted cells (Figure 2a). We have addressed this issue in transformed cells by Most importantly, 99% (964 of 967) of these differences studying the global gene expression patterns upon corresponded to increased transcription of each respec- knocking down MBDs using short hairpin RNA tive gene (Figure