Promoter Analysis of Epigenetically Controlled Genes in Bladder Cancer Srinivas Veerla, * Ioannis Panagopoulos, Yuesheng Jin, David Lindgren, and Mattias Ho ¨ glund Department of Clinical Genetics, Lund University Hospital, SE-22185, Lund, Sweden DNA methylation is an important epigenetic modification that regulates several genes crucial for tumor development. To iden- tify epigenetically regulated genes in bladder cancer, we performed genome wide expression analyses of eight-bladder cancer cell lines treated with the demethylating agents 5-aza-2 0 -cytidine and zebularine. To identify methylated C-residues, we sequenced cloned DNA fragments from bisulfite-treated genomic DNA. We identified a total of 1092 genes that showed 2- fold altered expression in at least one cell line; 710 showed up-regulation and 382 down-regulation. Extensive sequencing of promoters from 25 genes in eight cell lines showed an association between methylation pattern and expression in 13 genes, including both CpG island and non-CpG island genes. Overall, the methylation patterns showed a patchy appearance with short segments showing high level of methylation separated by larger segments with no methylation. This pattern was not associated with MeCP2 binding sites or with evolutionarily conserved sequences. The genes UBXD2, AQP11, and TIMP1 showed particular patchy methylation patterns. We found several high-scoring and evolutionarily conserved transcription factor binding sites affected by methylated C residues. Two of the genes, FGF18 and MMP11, that were down-regulated as response to 5-aza-2 0 -cytidine and zebularine treatment showed methylation at specific sites in the untreated cells indicating an activating result of methylation. Apart from identifying epigenetically regulated genes, including TGFBR1, NUPR1, FGF18, TIMP1, and MMP11, that may be of importance for bladder cancer development the presented data also highlight the organization of the modified segments in methylated promoters. This article contains Supplementary Material available at http://www. interscience.wiley.com/jpages/1045-2257/suppmat. V V C 2008 Wiley-Liss, Inc. INTRODUCTION Urothelial carcinoma, bladder cancer, is a com- plex disease associated with several genetic lesions of which activating mutations in FGFR3 and inacti- vating mutations of TP53 are the most common (Spruck et al., 1994; Cappellen et al., 1999; Bakkar et al., 2003). Urothelial carcinomas also show char- acteristic chromosomal alterations such as frequent loss of chromosome 9 and of chromosome arm 11p, and gain of chromosome 7 (Hoglund et al., 2001). Apart from genetic alterations, bladder cancers are also known to undergo epigenetic changes through altered methylation status of CpG-residues located in the vicinity of gene promoter regions. These changes generally result in gene silencing and sev- eral genes of importance for tumor development are known to undergo such changes e.g., CDKN1C and RUNX3 have recently been shown to be silenced by promoter methylation in bladder can- cer (Hoffmann et al., 2005; Kim et al., 2005). The altered DNA methylation pattern is maintained by a post-replicative mechanism that involves methyl- ation of hemi-methylated CpG by DNA methyl- tranferases (Bestor et al., 1992). Regions rich in CpG dinucleotides, CpG-islands (Bird, 1986), are, however, generally unmethylated when located in promoter regions of genes that are important for normal cell function (Jones and Laird, 1999; Baylin and Herman, 2000; Jones, 2002), hence, most CpG-island genes are in a hypomethylated state. De novo methylation of such regions results in a compact and silenced chromatin structure through binding methyl-DNA binding proteins and recruit- ment of histone deacetylases (Jones et al., 1998; Nan et al., 1998). Silencing of genes with CpG- poor promoters has also been shown to occur by methylation. In these cases modification of specific CpG residues is believed to affect the binding of regulatory proteins to their cognate binding sites (Mikovits et al., 1998; Bell and Felsenfeld, 2000; Hark et al., 2000; Bird, 2002). Even although pro- Supported by: Swedish Cancer Society, The Swedish Research Council, The Gunnar Nilsson Cancer Foundation, The Crafood Foundation. *Correspondence to: Srinivas Veerla, Department of Clinical Genetics, Lund University Hospital, SE-22185, Lund, Sweden. E-mail: srinivas.veerla@med.lu.se Received 5 November 2007; Accepted 17 December 2007 DOI 10.1002/gcc.20542 Published online 14 January 2008 in Wiley InterScience (www.interscience.wiley.com). V V C 2008 Wiley-Liss, Inc. GENES, CHROMOSOMES & CANCER 47:368–378 (2008)