Silencing of Bidirectional Promoters by DNA Methylation in Tumorigenesis Jingmin Shu, Jaroslav Jelinek, Hao Chang, Lanlan Shen, Taichun Qin, Woonbok Chung, Yasuhiro Oki, and Jean-Pierre J. Issa Department of Leukemia, M.D. Anderson Cancer Center, Houston, Texas Abstract CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563 , and KCNK15/ BF195580 . Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2¶-deoxycytidine treatment reac- tivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down- regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter- associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer. (Cancer Res 2006; 66(10): 5077-84) Introduction Bidirectional gene organization is defined as two genes arranged head to head on opposite strands with <1,000 bp between their transcription start sites (1). Generally, about 20% of genes are organized in this way (1, 2). It was previously suggested that bidirectional organization may provide stronger resistance to invasion by transposable elements (2), which is possibly one of the reason why many important genes (e.g., 30% of housekeeping genes) are arranged in this way (1). Remarkably, bidirectional promoters are particularly common in DNA repair genes with a frequency of 40% (1). It remains to be determined if this is also true for tumor suppressor genes. The GC content of bidirectional promoters was reported to be higher than that of regular promoters (3), and it was proposed that these promoters could be more resistant to methylation for protection of essential genes. Like other promoters, bidirectional promoters are also frequently associated with CpG islands (2). A CpG island is a short region of DNA in which the frequency of the CG sequence is less suppressed (4, 5). Hypermethylation of CpG islands in promoter regions usually results in gene silencing, and several tumor suppressor genes are hypermethylated in their promoter regions in cancers, which is thought to contribute to tumorigen- esis (3, 6, 7). Most bidirectional promoters coordinately regulate transcription of the gene pair (3), but it remains to be determined whether hypermethylation of CpG islands in such promoters is also able to silence genes in both directions. If this were true, silencing of bidirectional promoters by hypermethylation would possibly be an important mechanism for oncogenesis because a single ‘‘hit’’ within these promoters could potentially disable two tumor suppressor genes simultaneously, which could accelerate tumor development, according to the multiple hit theory of tumorigenesis (8). Here, we sought to determine the effect of hypermethylation of bidirectional promoters on the expression of either gene in the gene pair and study the likelihood of this event in cancer. We report on three bidirectional promoters hypermethylated in cancer, with silencing of both genes in each case. Our data expand the effects of CpG island methylation in tumor development. Materials and Methods Cell lines and tissues. Cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA). All of the patient samples, both cancerous and normal, were obtained from an estab- lished tissue bank at the University of Texas M.D. Anderson Cancer Center (Houston, TX). Patients gave informed consent for the collection of residual tissue as per institutional guidelines. The studies were approved by the institutional review board of the M.D. Anderson Cancer Center. 5¶-Rapid amplification of cDNA ends PCR. Human fetal brain marathon-ready cDNA (Clontech, Mountain View, CA) was used as template for PCR according to the manufacturer’s instruction with primers listed in Supplementary Table S1. The largest-size PCR products were gel purified by the QIAprep Gel Purification kit (Invitrogen, Carlsbad, CA) and cloned into a pCR 4.0-TOPO vector (Invitrogen). Individual clones were sequenced at the DNA sequencing core facility at the University of Texas M.D. Anderson Cancer Center. DNA bisulfite treatment. After DNA extraction, DNA was treated with bisulfite as reported previously (9). Two micrograms of genomic DNA were denatured by 0.2 mol/L NaOH at 37jC for 10 minutes followed by incubation with freshly prepared 30 AL of 10 mmol/L hydroquinone and 520 AL of 3 mol/L sodium bisulfite (pH 5) at 50jC for 16 hours. DNA was purified with a Wizard miniprep Column (Promega Co., San Diego, CA), desulfonated with 0.3 mol/L NaOH at 25jC for 5 minutes, precipitated with ammonium acetate and ethanol, and resuspended in 30 AL distilled water. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Jean-Pierre J. Issa, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Unit 428, 1515 Holcombe, Houston, TX 77030. Phone: 713-745-2260; Fax: 713-745-1683; E-mail: jpissa@mdanderson.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2629 www.aacrjournals.org 5077 Cancer Res 2006; 66: (10). May 15, 2006 Research Article Research. on June 25, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from