Masahiko Shiraishi

HOX gene clusters are hotspots of de novo methylation in CpG islands of human lung adenocarcinomas

CpG island methylation results in the silencing of the associated gene and is an important step in tumorigenesis. Following a comprehensive isolation of CpG islands that were methylated in human lung adenocarcinoma, we found that in cancer cells de novo CpG island methylation generally occurred in a sporadic manner. However, some methylated CpG islands appeared to cluster in discrete chromosomal regions. In this study, we have investigated the methylation status of CpG islands located at such chromosomal loci. We have found that many CpG islands at the HOXA and HOXD loci were methylated in human lung adenocarcinoma. The de novo methylation of these CpG islands was also observed in patients DNA from noncancerous portions of lung tissue. These results indicate the presence of specific chromosomal regions that are susceptible to de novo methylation.

An overview of the analysis of DNA methylation in mammalian genomes.

Abstract DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5- methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.

A comprehensive catalog of CpG islands methylated in human lung adenocarcinomas for the identification of tumor suppressor genes

CpG island methylation is an important mechanism in gene silencing and is a key epigenetic event in cancer development. As yet, the number and identities of the genes that are inactivated in cancer cells has not been determined. In order to address this issue, we have performed a comprehensive isolation of CpG islands that are methylated in human lung adenocarcinomas. We have isolated approximately 200 CpG islands that are methylated in tumor DNA including those of known tumor-associated genes such as the HOXA5 gene. As the library contains the CpG islands of a number of known tumor suppressor genes it is highly likely that additional, previously unidentified tumor suppressor genes, will be present. On average, 1–2% of CpG islands were methylated specifically in tumors although this figure differed greatly between patients. This study provides an important resource in the search for genes inactivated in tumors and for the investigation of epigenetic dysregulation of gene expression by CpG island methylation.

Bisulfite modification for analysis of DNA methylation.

Bisulfite is known to deaminate cytosine in nucleic acids, while 5-methylcytosine resists this bisulfite action. For this reason, bisulfite treatment has been used for detecting 5-methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as playing an important role in the control of gene function. This procedure, called bisulfite genomic sequencing, is a principal method for the analysis of DNA methylation in various biological phenomena, including human diseases such as cancer. This unit describes an efficient procedure utilizing a newly developed high-concentration bisulfite solution. Protocols for this methodology are supplemented with discussions focused on chemical aspects of the bisulfite treatment.

Tight interaction between densely methylated DNA fragments and the methyl-CpG binding domain of the rat MeCP2 protein attached to a solid support.

Abstract In a previous report we have found that a number of short DNA fragments methylated at CpG sequences bound more tightly to a methyl-CpG binding column than DNA fragments having a larger number of methyl- CpG sequences. The column consists of a polypeptide comprising the DNA binding domain of the rat MeCP2 protein attached to a solid support. In the present study, we have investigated the features of short DNA fragments which bind tightly to a methyl-CpG binding column. Tight binding was observed when the DNA fragment had a high density of methyl-CpG sequences. Many of these fragments, derived from human genomic DNA, contained Alu repeated sequences supporting the previous observation that the highly-abundant Alu sequences are highly methylated. Our results suggest that methyl-CpG density is an important factor in the interaction between DNA fragments and the DNA binding domain of MeCP2 attached to a solid support.

Absence of Activating Mutations in the Transmembrane Domain of the c-erbB-2 Proto-oncogene in Human Lung Cancer

The rat neu gene is known to be activated by a point mutation in its predicted transmembrane domain. Overexpression of the human homologue of neu, the c‐erbB‐2 gene, in human lung cancer has been reported, and a similar activating point mutation has been suggested. Therefore, we tested for possible aberrations of the c‐erbB‐2 gene in the region of the transmembrane domain in surgical specimens of human primary lung cancer from 190 patients, and also examined 24 metastases and 26 specimens of noncancerous portions of the lung of the same patients. Single‐strand conformation polymorphism analysis of polymerase chain reaction products revealed no point mutations in the target domain in any of these specimens.

Alteration of mosaic methylation of the repeat unit of the human ribosomal RNA genes in lung cancer.

Abstract We have investigated the methylation status of the repeat unit of the human ribosomal RNA genes in lung cancer. Using a Southern blot analysis approach we have determined that the non-transcribed region of these genes was generally heavily methylated, while the transcribed region was not methylated in either tumor or normal DNA. Our study also revealed that, in one tumor, the boundary of mosaic methylation of the repeat unit was not distinct. In the same tumor, both the non-transcribed ribosomal spacer region and the L1 interspersed repeat sequences became partially demethylated. In tumor cells, the methylation status of DNA can be altered, but the methylation of subtelomeric repeats was found to be maintained. These results suggest that the mosaic methylation of the repeat unit is not necessarily maintained in tumor DNA, while subtelomeric repeats escape tumor-specific wave of demethylation.

Treatment of tumor cells with histone deacetylase inhibitors results in altered recruitment of methyl-CpG binding proteins to a methylated CpG island.

Abstract When human cancer cells with silencing of the CDH1 gene associated with CpG island methylation and histone deacetylation were treated with histone deacetylase inhibitors, alteration in recruitment of methyl-CpG binding proteins (MBPs) to the methylated CDH1-CpG island was observed, as well as altered histone acetylation status. This change was independent of the histone deacetylase inhibitor used. These results suggest that histone hyperacetylation provides a more open chromatin structure conformation for the recruitment of additional MBPs.

Segregation of partly melted molecules: isolation of CpG islands by polyacrylamide gel electrophoresis.

Abstract The technique of segregation of partly melted molecules (SPM) is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The approach is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. The SPM methodology has successfully been applied to the identification of genes from anonymous, unsequenced DNA fragments and CpG islands methylated in human cancer. In this article the theoretical background and practical application of the SPM method is reviewed.

Segregation of partly melted DNA molecules.

Segregation of partly melted DNA molecules is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The method stands on the observation that the electrophoretic mobility of partly melted DNA fragments in a denaturing gradient gel is low and that they persist in the gel so long as the remaining helical part is sufficiently resistant to strand dissociation and dissociates slowly. Such features are observed in DNA fragments derived from CpG islands. These DNA fragments are preferentially retained in a denaturing gradient gel after prolonged electric field exposure, permitting the enrichment of DNA fragments derived from CpG islands. The principle and practical application of this method are reviewed.