Genome-wide profiling of promoter methylation in human

Abstract

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer

Comparative isoschizomer profiling of cytosine methylation: The HELP assay

“…The signals from individual HpaII-amplifiable fragments in (G+C)-rich and -poor regions are otherwise directly comparable. In addition, the use of a methylation-insensitive isoschizomer controls for copy number, relative difficulty with PCR amplification of different loci, and polymorphisms at the restriction enzyme cleavage site, as was demonstrated in a recent publication (Hatada et al 2006).…”

Ligand-Activated PPARα-Dependent DNA Demethylation Regulates the Fatty Acid β-Oxidation Genes in the Postnatal Liver

“…Human fetal (male 18 and 22 weeks and female 18 weeks after fertilization) and adult (male 20, 44, and 59 years old) liver genomic DNAs were purchased from BioChain Institute Inc. (Newark, CA). The Microarray-Based Integrated Analysis of Methylation by Isoschizomers (MIAMI) analysis, a genomewide analysis of DNA methylation using a gene promoter array and methylation-sensitive restriction enzyme, was performed as described (20)(21)(22). Detailed experimental procedure is described in the Supplementary Data.…”

Gene expression changes in normal haematopoietic cells

“…87 By changing the DNA sequence, investigators were able to design primers or hybridization probes that differentially target non-modified (methylated) and modified (unmethylated) sequences, leading to the development of global methylation arrays. [87][88][89] Currently, there are a number of techniques that can be used to examine the relationship of chromatin structure and genes, but the workhorse of these investigations is the chromatin immunoprecipitation (CHiP) assay. 90 With CHiP assays, chromatin is precipitated with an antibody, and the pellet is interrogated by a variety of methods, including PCR of specific genomic fragments of interest, and as with methylation arrays, high throughput CHiP arrays are becoming increasingly more comprehensive and popular in the research community.…”