Research Article MIRA-Assisted Microarray Analysis, a New Technology for the Determination of DNA Methylation Patterns, Identifies Frequent Methylation of Homeodomain-Containing Genes in Lung Cancer Cells Tibor Rauch,1 Hongwei Li,1 Xiwei Wu,2 and Gerd P. Pfeifer1 Divisions of 1Biology and 2Biomedical Informatics, Beckman Research Institute of the City of Hope, Duarte, California Abstract hypermethylation generally leads to inactivation of gene expres- We present a straightforward and comprehensive approach sion, this epigenetic alteration is considered to be a key mechanism for DNA methylation analysis in mammalian genomes. The for long-term silencing of tumor suppressor genes. The importance methylated-CpG island recovery assay (MIRA), which is based of promoter methylation in functional inactivation of lung cancer on the high affinity of the MBD2/MBD3L1 complex for suppressor genes is becoming increasingly recognized. It is methylated DNA, has been used to detect cell type–dependent estimated that between 0.5% and 3% of all genes carrying CpG- differences in DNA methylation on a microarray platform. The rich promoter sequences (so-called CpG islands) may be silenced procedure has been verified and applied to identify a series of by DNA methylation in lung cancer (1, 11). This means that there novel candidate lung tumor suppressor genes and potential are most likely several hundred genes that are incapacitated by this DNA methylation markers that contain methylated CpG pathway. Some of these genes may be bona fide tumor suppressor islands. One gene of particular interest was DLEC1, located genes, but in other cases, the methylation event may be a at a commonly deleted area on chromosome 3p22-p21.3, consequence of gene silencing or may somehow be associated with which was frequently methylated in primary lung cancers and tumor formation rather than being a cause of tumorigenesis. melanomas. Among the identified methylated genes, homeo- Several specific genes are methylated in lung cancer, including CDKN2A, RASSF1A, RARb, MGMT, GSTP1, CDH13, APC, DAPK, domain-containing genes were unusually frequent (11 of the TIMP3 top 50 hits) and were targeted on different chromosomes. , and several others (12–16). The methylation frequency These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, (the percentage of tumors analyzed that carry methylated alleles) HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show ranges from <10% to >80% but these numbers differ widely that MIRA-assisted microarray analysis has a low false- depending on the histologic type of tumor, the study population, positive rate and has the capacity to catalogue methylated and/or the methods used to assess methylation. To improve the CpG islands on a genome-wide basis. The results support the sensitivity of screening tools for the detection of early lung cancer, hypothesis that cancer-associated DNA methylation events DNA methylation markers have shown great promise (7, 17, 18). do not occur randomly throughout the genome but at least However, many more markers that could have improved specificity some are targeted by specific mechanisms. (Cancer Res 2006; in discriminating tumor from normal tissue and are methylated 66(16): 7939-47) at a high frequency in lung tumors have likely not yet been discovered. Introduction To analyze DNA methylation patterns on a genome-wide scale, several techniques have been developed, but none of them has yet In mammalian cells, the DNA base 5-methylcytosine occurs at ¶ reached wide acceptance. Most methods currently available are 5 -CpG dinucleotides and provides the basis for a common mode of labor-intensive and use methylation-sensitive restriction endonu- epigenetic inheritance. Changes in DNA methylation patterns cleases, and thus are limited by the occurrence of the respective occur in a developmental stage– and tissue-specific manner and sites within the target sequence. Another way to find methylated often accompany tumor development, most notably in the form of genes is by using expression microarrays to identify genes CpG island hypermethylation (1–10). During tumorigenesis, both reactivated by treatment with DNA methylation inhibitors (e.g., alleles of a tumor suppressor gene need to be inactivated by geno- ‘5-aza-deoxycytidine; refs. 19–21). This approach is effective but mic changes such as chromosomal deletions or loss-of-function can only be used with cell lines. Recently, genomic tiling and BAC mutations in the coding region of a gene. As an alternative microarrays have been introduced to map methylation patterns mechanism, transcriptional silencing by hypermethylation of CpG (22, 23). These methods are also limited, both in terms of their level islands spanning the promoter regions of tumor suppressor genes of resolution and in terms of the requirements for restriction is a common and important process in carcinogenesis. Because endonuclease recognition sites. An antibody against 5-methylcyto- sine has been used in immunoprecipitation experiments combined with microarrays (6, 23). However, this antibody requires ssDNA for recognition, which is sometimes difficult to achieve in CpG-rich Note: Supplementary data for this article are available at Cancer Research Online DNA regions. Here we describe a new genome-wide DNA (http://cancerres.aacrjournals.org/). Requests for reprints: Gerd P. Pfeifer, Division of Biology, Beckman Research methylation detection method that depends neither on restriction Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010. Phone: 626- endonucleases nor on specific antibodies. This method is based on 301-8853; Fax: 626-358-7703; E-mail: [email protected]. I2006American Association for Cancer Research. the methylated-CpG island recovery assay (MIRA), which we doi:10.1158/0008-5472.CAN-06-1888 previously applied for testing the methylation status of specific www.aacrjournals.org 7939 Cancer Res 2006; 66: (16). August 15, 2006 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research genes (24). It makes use of the high affinity of the MBD2b/MBD3L1 log 2 ratios between the dye swap pairs. Based on our experience, the complex for methylated DNA (24, 25). Here we show that MIRA can combined Lowess and dye swap normalization approach can best reduce be used to analyze the DNA methylation status of a large number of variability. CpG island methylation profiles were determined by ratios genes simultaneously using a microarray approach. between MIRA-enriched and unenriched samples (enrichment factor) for both tumor and normal tissues. The ratios of the enrichment factors between cancer and normal DNA samples will measure the methylation Materials and Methods difference between cancer and normal tissue. To identify the CpG islands MIRA and microarray analysis. DNA obtained from normal human that are differentially methylated between normal and tumor cell DNA, bronchial epithelial (NHBE) cells and from the lung cancer cell line A549 methylation profiles were compared using statistical linear model in was digested with MseI(5¶-TTAA), which produces small (f200-300 bp) LIMMA. For target gene selection, unadjusted P values were set at a level fragments and generally cuts outside of CpG islands. Linkers (upper of 0.05, and the fold change between cancer MIRA/Input versus normal strand sequence, 5¶-TAGAATTCAGATCTCCCG-3¶; lower strand sequence, MIRA/Input (difference factor) was set at >2. Direct comparison of MIRA- 3¶-CTTAAGTCTAGAGGGCCCAGTGGCG-5¶) were ligated to the MseI- enriched fractions from tumor and normal tissue DNA provided digested DNA and enrichment of the methylated fraction was done by independent confirmation for the methylation differences observed MIRA as previously described (24). Briefly, 1 Ag of purified GST-tagged although the latter analysis may be affected by differences in gene copy MBD2b protein and 1 Ag of purified His-tagged MBD3L1 protein were numbers between normal and tumor tissue. preincubated and bound to a glutathione sepharose CL-4B matrix DNA methylation analysis using combined bisulfite restriction (Amersham Biosciences, Piscataway, NJ). The plasmids used to produce analysis and bisulfite sequencing. DNA was isolated from cell lines or these proteins are available on request. This matrix was incubated with 500 frozen tumors and matched normal tissue by standard phenol-chloroform ng of MseI-cut and linker-ligated genomic DNA in 400 AL of a binding extraction and ethanol precipitation. Non–small-cell lung carcinoma tumor reaction mixture [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1 mmol/L tissue samples and matching normal tissues removed with surgery were EDTA, 1 mmol/L DTT, 3 mmol/L MgCl2, 0.1% Triton-X100, 5% glycerol, obtained from the frozen tumor bank of the City of Hope National Medical 25 Ag/mL bovine serum albumin, and 1.25 Ag/mL sonicated JM110 (dcm Center. The combined bisulfite restriction analysis (COBRA) assays were minus) bacterial DNA] for 240 minutes at 4jC on a rocking platform. After done using the method of Xiong and Laird (28). DNA was treated with washing the pelletted sepharose beads thrice with binding buffer containing sodium bisulfite and purified as described (24). PCR primers for 700 mmol/L NaCl, the methylated DNA–enriched genomic DNA fraction amplification of specific targets in bisulfite-treated DNA are listed in was eluted by addition of guanidinium hydrochloride–containing buffer and Supplementary Table S1. For sequence analysis, the PCR products