DNA METHYLATION AND GENOME FUNCTION PROJECT

18.DNA METHYLATION AND GENOME FUNCTION PROJECT

    The research of the DNA Methylation and Genome Function Project is aimed at the elucidation of epigenetic processes in carcinogenesis. In particular, these studies concern mechanisms associated with CpG islands as CpG island methylation is a common epigenetic aberration in cancer. For this purpose, comprehensive isolation of CpG islands methylated in cancer and investigation of the molecular mechanism of gene silencing by CpG island methylation have been carried out.
Comprehensive Isolation of CpG Islands Methylated in Human Adenocarcinomas of the Lung

    Segregation of partly melted molecules (SPM) is a convenient and efficient method for the isolation of CpG island-associated DNA fragments based on their retention by denaturing gradient gel electrophoresis (DGGE). DGGE is a technique employed to separate DNA fragments on the basis of local variation in base composition within the DNA fragments. This separation depends on the markedly reduced electrophoretic mobility that occurs when part of a DNA fragment melts resulting in a structure that is partly helical and partly random chain. On prolonged electric field exposure, the retarded fragments will fade away through strand dissociation. If the stability of the helical part is appreciably higher than that of the melted part, the dissociation rate will be low and the retarded, partly melted molecule remains in the gel for several hours. It would be reasonable to speculate that DNA fragments derived from the edges of CpG islands consist of at least two different melting domains since the G+C-rich nature of the CpG island sequence results in high Tm, while flanking non-island sequences are not G+C-rich and would be lower melting. As a consequence, preferential retention of DNA fragments derived from the edges of CpG islands after prolonged field exposure is predicted. Using the SPM method, CpG islands from human chromosomal region 11q13 were successfully isolated. An independent technique, methylated-DNA-binding column (MBD column) chromatography, permits separation of DNA fragments on the basis of the number of methyl-CpG sequences in the fragment, and it allows the separation of methylated CpG islands from those that are not methylated. SPM and MBD column chromatography were combined to isolate CpG islands that are methylated in human adenocarcinomas of the lung.
    Highly methylated DNA fragments in cancer were enriched by MBD column chromatography and used for construction of a library. Cloned DNA fragments were analyzed by the SPM method to isolate DNA fragments associated with CpG islands. CpG islands methylated in cancer were grouped into two classes;those specifically methylated in cancer cells (Class I) and those methylated in cancer cells as well as in normal somatic tissues (Class II). Several CpG islands reported to be methylated in cancer were isolated from the library as Class I islands. The presence of these CpG islands in the library strongly suggests that CpG island fragments associated with novel tumor suppressor genes may also be present in the library. Some CpG islands associated with human orthologues of the known imprinted genes in mice were isolated as Class II islands. The methylated CpG island library will be an invaluable tool for obtaining a clearer picture of aberrant CpG island methylation and elucidating the epigenetic process in carcinogenesis.
Molecular Mechanism of Gene Silencing by CpG Island Methylation

    The methylation status of the CpG island of the human E-cadherin (CDH1) gene in cancer cell lines has been analyzed by MBD column chromatography. DNA fragments containing this island showed different affinities to the column among silenced cell lines, where the CDH1 gene is not expressed. Bisulfite genomic sequencing analysis revealed that high affinity to the column corresponded to high methylation of the island, while low affinity corresponded to low density methylation. In one repressed cell line, the promoter region was poorly methylated while methyl-CpGs were distributed relatively highly to the 3'region of the CpG island. Despite these differences, chromatin structures of the promoter region were commonly compacted, and transcription factors were excluded from their binding sites in silenced cell lines. Using chromatin immunoprecipitation analysis, it was shown that histones associated with the CpG island of the CDH1 gene were also differentially acetylated among silenced cell lines and that the binding of methyl-CpG binding proteins was also heterogeneous. These results suggest that high density methylation or promoter methylation is not always necessary for gene silencing by CpG island methylation and that heterogeneity exists in methylation and histone acetylation. Elucidation of the molecular mechanisms responsible for these phenomena is currently in progress.




18.DNA METHYLATION AND GENOME FUNCTION PROJECT



	The research of the DNA Methylation and Genome Function Project is aimed at the elucidation of epigenetic processes in carcinogenesis. In particular, these studies concern mechanisms associated with CpG islands as CpG island methylation is a common epigenetic aberration in cancer. For this purpose, comprehensive isolation of CpG islands methylated in cancer and investigation of the molecular mechanism of gene silencing by CpG island methylation have been carried out. Comprehensive Isolation of CpG Islands Methylated in Human Adenocarcinomas of the Lung Segregation of partly melted molecules (SPM) is a convenient and efficient method for the isolation of CpG island-associated DNA fragments based on their retention by denaturing gradient gel electrophoresis (DGGE). DGGE is a technique employed to separate DNA fragments on the basis of local variation in base composition within the DNA fragments. This separation depends on the markedly reduced electrophoretic mobility that occurs when part of a DNA fragment melts resulting in a structure that is partly helical and partly random chain. On prolonged electric field exposure, the retarded fragments will fade away through strand dissociation. If the stability of the helical part is appreciably higher than that of the melted part, the dissociation rate will be low and the retarded, partly melted molecule remains in the gel for several hours. It would be reasonable to speculate that DNA fragments derived from the edges of CpG islands consist of at least two different melting domains since the G+C-rich nature of the CpG island sequence results in high Tm, while flanking non-island sequences are not G+C-rich and would be lower melting. As a consequence, preferential retention of DNA fragments derived from the edges of CpG islands after prolonged field exposure is predicted. Using the SPM method, CpG islands from human chromosomal region 11q13 were successfully isolated. An independent technique, methylated-DNA-binding column (MBD column) chromatography, permits separation of DNA fragments on the basis of the number of methyl-CpG sequences in the fragment, and it allows the separation of methylated CpG islands from those that are not methylated. SPM and MBD column chromatography were combined to isolate CpG islands that are methylated in human adenocarcinomas of the lung. Highly methylated DNA fragments in cancer were enriched by MBD column chromatography and used for construction of a library. Cloned DNA fragments were analyzed by the SPM method to isolate DNA fragments associated with CpG islands. CpG islands methylated in cancer were grouped into two classes;those specifically methylated in cancer cells (Class I) and those methylated in cancer cells as well as in normal somatic tissues (Class II). Several CpG islands reported to be methylated in cancer were isolated from the library as Class I islands. The presence of these CpG islands in the library strongly suggests that CpG island fragments associated with novel tumor suppressor genes may also be present in the library. Some CpG islands associated with human orthologues of the known imprinted genes in mice were isolated as Class II islands. The methylated CpG island library will be an invaluable tool for obtaining a clearer picture of aberrant CpG island methylation and elucidating the epigenetic process in carcinogenesis. Molecular Mechanism of Gene Silencing by CpG Island Methylation The methylation status of the CpG island of the human E-cadherin (CDH1) gene in cancer cell lines has been analyzed by MBD column chromatography. DNA fragments containing this island showed different affinities to the column among silenced cell lines, where the CDH1 gene is not expressed. Bisulfite genomic sequencing analysis revealed that high affinity to the column corresponded to high methylation of the island, while low affinity corresponded to low density methylation. In one repressed cell line, the promoter region was poorly methylated while methyl-CpGs were distributed relatively highly to the 3'region of the CpG island. Despite these differences, chromatin structures of the promoter region were commonly compacted, and transcription factors were excluded from their binding sites in silenced cell lines. Using chromatin immunoprecipitation analysis, it was shown that histones associated with the CpG island of the CDH1 gene were also differentially acetylated among silenced cell lines and that the binding of methyl-CpG binding proteins was also heterogeneous. These results suggest that high density methylation or promoter methylation is not always necessary for gene silencing by CpG island methylation and that heterogeneity exists in methylation and histone acetylation. Elucidation of the molecular mechanisms responsible for these phenomena is currently in progress.