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

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.     

High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80-100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.     

Hepatitis B viral DNA is methylated in liver tissues

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.     

Active gene sequences are undermethylated.

The degree of methylation of active regions of the chromosome has been investigated by several techniques. DNase I (deoxyribonuclease I, EC 3.1.21.1) was used to introduce nicks in the active regions of the nucleus and thereby specifically label these areas. By using the methylation-specific restriction enzymes Hpa II and Hha I it could be shown that active genes are more sensitive to these probes than are other parts of the genome. In order to measure the amount of methylation at all CpG residues, DNA was nick-translated in the presence of [alpha-32P]dGTP as the sole nucleotide source and the methylated cytosine was detected by the standard nearest-neighbor analysis. Using this assay, we found that about 70% of all CpG sequences in animal cell DNA are methylated. In active nuclear regions that are sensitive to DNase I, only 30-40% of the CpG residues are methylated. This method was also employed to study the gene sequences that are complementary to cellular RNA. By this criterion expressed gene sequences are only 20-30% methylated. These data suggest that undermethylation is a general phenomenon in all actively transcribed genes.     

Predicting aberrant CpG island methylation

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.     

Pattern of methylation of two genes coding for housekeeping functions.

The distribution of sites that can be methylated was analyzed in the Chinese hamster adenine phosphoribosyl-transferase (aprt) gene and the patterns of methylation of this gene and the mouse dihydrofolate reductase (dhfr) gene were studied by using CpG restriction enzymes. Both genes were found to be unmethylated completely at their 5'-end region and methylated heavily throughout the rest of the gene. Because the hamster aprt gene can be inhibited by DNA methylation in vivo, the results suggest that 5' undermethylation of this gene may be a necessary condition for its expression. The pattern of methylation of each of these two genes was similar in sperm and all other somatic tissue DNAs. This is in contrast to many tissue-specific genes that were found to be highly methylated in sperm DNA and undermethylated in the tissue in which they are expressed. This result is consistent with the fact that both aprt and dhfr are key enzymes in the biosynthesis of nucleotides and therefore expected to be synthesized in all cells.     

Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression

Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9-25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., Held, W.A., Yoshida, S. & Nagase, H. (2003) Nucleic Acids Res. 31, 4490-4496]. Analysis of 14 TDMs by methylation-specific PCR and by bisulfite genomic sequencing confirms that the regions identified by RLGS are differentially methylated in a tissue-specific manner. The results indicate that 5% or more of the CpG islands are TDMs, disputing the general notion that all CpG islands are unmethylated. Some of the TDMs are within 5' promoter CpG islands of genes, which exhibit a tissue-specific expression pattern that is consistent with methylation status and a role in tissue differentiation.     

Aberrant DNA methylation profile of chronic and transformed classic Philadelphia-negative myeloproliferative neoplasms

Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.     

Epigenomic analysis of Alu repeats in human ependymomas

Global loss of DNA methylation has been known for decades as an epigenomic aberration associated with carcinogenesis and cancer progression. Loss of DNA methylation affects predominantly repetitive elements, which encompass >50% of the CpG dinucleotides present in the human genome. Because of the lack of an effective approach, no studies have been conducted to reveal such genome-wide methylation changes at a single-base resolution. To precisely determine the CpG sites with methylation loss during progression of pediatric intracranial ependymomas, we exploited a high-throughput bisulfite sequencing approach that simultaneously generates methylation profiles for thousands of Alu elements and their flanking sequences. Comparison of the methylation profiles of normal and tumor tissues revealed that the methylation status of the majority of CpG sites adjacent to or within Alu repeats remain unaltered, while a small set of CpG sites gain or lose methylation in ependymomas. Compared to the CpG sites with stable methylation level between normal control and ependymomas, the differentially methylated CpG sites are enriched in the sequences with low CpG density in the flanking regions of Alu repeats, rather than within the Alu sequences themselves. In addition, the CpG sites that are hypermethylated in ependymomas are proximal to CpG islands, whereas those that are hypomethylated are overrepresented in intergenic regions. Lastly, aberrant methylation of several genomic loci was confirmed to be associated with the aggressive primary tumors and the relapsed ependymomas.