Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay

Abstract

De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX, LHX, PAX, DLX, and Engrailed, were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7- and HOXA9-associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.

Fig. 1.

Analysis of the HOXA cluster region on chromosome 7 with ENCODE NimbleGen tiling arrays. The analysis covers a 155-kb region. DNA from the GM06990 cell line was used for three independent MIRA reactions by using the amplification and labeling procedure as described in Materials and Methods. (Upper) The signal ratio of MIRA-enriched versus input DNA is plotted along the chromosome. The green boxes indicate the 39 CpG islands (see SI Table 1). The numbering refers to HOXA gene number, and the arrows indicate the direction of transcription. A COBRA using BstUI digestion (see Materials and Methods) was conducted for the regions indicated to confirm the methylation data obtained by MIRAs. Some of the BstUI cleavage fragments are too small to be visible on the gels. −, to control digestion with no BstUI; +, BstUI-digested samples.

Fig. 2.

DNA and histone methylation profile at HOXA cluster genes in GM06990 cells. (A) DNA and histone methylation profile analysis of the entire HOXA cluster in GM06990 cells. The MIRA-enriched methylated DNA fraction and input fraction were mixed and hybridized onto NimbleGen ENCODE arrays. Note that in this experiment, the MIRA-enriched DNA and the input DNA were processed without amplification and were directly labeled and hybridized to the array to make these data methodologically comparable with the ChIP data. ChIP with anti-histone H3K4m3 antibody was performed, and the DNA was hybridized onto Sanger ENCODE3.1.1 DNA microarrays (data were obtained from the ENCODE database). The pink shading indicates the promoter-associated CpG islands. (B) (Upper) DNA and histone methylation and mRNA expression profile analysis of the HOXA5 and HOXA6 genes. Data for H3K4 methylation and mRNA expression are from the ENCODE database. (Lower) Bisulfite sequencing verification of the DNA methylation status of the indicated CpG island regions.

Fig. 3.

DNA methylation profiles of chromosomes 2, 7, 12, and 17 that carry the HOXA, HOXB, HOXC, and HOXD gene clusters. MIRA-enriched fractions from A549 lung cancer cells and from NHBE cells were mixed and hybridized onto Agilent CpG island arrays. The ratio (fold difference) plotted indicates individual probe signals (blue diamonds) for hypermethylated CpG islands in A549 cells (A549/NHBE signal ratio). The numbers indicate selected areas of densely hypermethylated CpG island clusters for the genes indicated at the bottom.

Fig. 4.

Methylation of HOXA genes in normal lung tissue and matching stage 1 squamous cell carcinoma samples. (A) Methylation differences between squamous cell carcinomas and matching normal pairs (pairs 1–4) were detected by COBRAs of HOX gene targets. T, tumor; N, normal tissues; −, control digestion with no BstUI; +, BstUI-digested samples. (B) Summary of the methylation status of promoters in the HOXA gene cluster.