DNA METHYLATION AND GENOME FUNCTION PROJECT

19.DNA METHYLATION AND GENOME FUNCTION PROJECT

    Research in the DNA Methylation and Genome Function Project is focused on the relationship between the modification of chromatin components and gene activities in cancer cells. DNA and histones are major components of the nucleosome, a basic unit of chromatin. Active and inactive chromatins differ in modification of these components. DNA methylation and histone acetylation and/or methylation at a specific chromosomal locus alters the chromatin structure by recruiting regulatory proteins that interact with the specific modification. The elucidation of the functional relationship between these modifications is the major research subject in this project. Development of novel experimental techniques for this purpose is an additional important research subject ^(242-²⁴⁴⁾.
The Comprehensive Isolation of CpG Islands Methylated in Human Lung Cancer

    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, a comprehensive isolation of CpG islands that are specifically methylated in human lung adenocarcinomas has been performed ⁽²⁴⁵⁾. Approximately 200 CpG islands that are methylated in tumor DNA has been isolated. These CpG islands include 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 the epigenetic dysregulation of gene expression by CpG island methylation.
    Following a comprehensive isolation of CpG islands that were methylated in human lung adenocarcinoma, it was shown 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. Many CpG islands at the HOXA and HOXD loci were methylated in human lung adenocarcinoma ⁽²⁴⁶⁾. The de novo methylation of these CpG islands was also observed in patient’s DNA from noncancerous portions of lung tissue. These results indicate the presence of specific chromosomal regions that are susceptible to de novo methylation.
Heterogeneity in the Modification of Chromatin Components in Cancer Cells

    The structural alteration of chromatin has a key role in regulating gene expression. The alteration of chromatin is mediated by modification of its components. A detailed understanding of the relationship between these modifications, notably, methylation of the full-length CpG island, the association of methyl-CpG binding proteins (MBPs), and the acetylation and methylation of histones in gene silencing is vitally important. Currently however, the manner in which chromatin components, associated with a specific gene, are modified is poorly understood. The manner of the differential association between CpG methylation, MBPs, and histone modification in the entire CpG island of the human E-cadherin (CDH1) gene in cancer cells have been investigated ⁽²⁴⁷⁾. Of the cell lines with CDH1 transcriptional repression, the distribution of methyl-CpGs in the CpG island differed markedly. In a cell line with gene silencing, the promoter region was almost methylation-free. Chromatin immunoprecipitation analysis revealed that the acetylation status of histone H4 differed between cell lines. However, deacetylated histone H3 was associated with the CpG island in all silenced cell lines. Binding of MeCP2 was also detected in all silenced cell lines. Additional binding of the MBD1 protein was detected in a cell line in which the promoter region was poorly methylated and only histone H3 was deacetylated. Binding of MBD2 protein was detected in all other silenced cell lines. Histone H3 lysine 9 was methylated in all silenced cells, while histone H3 lysine 4 was methylated in some silenced cell lines. These results demonstrate that chromatin components associated with inactive CDH1 chromatin are heterogeneously modified and suggests the presence of multiple pathways for the formation of inactive chromatin.




19.DNA METHYLATION AND GENOME FUNCTION PROJECT



	Research in the DNA Methylation and Genome Function Project is focused on the relationship between the modification of chromatin components and gene activities in cancer cells. DNA and histones are major components of the nucleosome, a basic unit of chromatin. Active and inactive chromatins differ in modification of these components. DNA methylation and histone acetylation and/or methylation at a specific chromosomal locus alters the chromatin structure by recruiting regulatory proteins that interact with the specific modification. The elucidation of the functional relationship between these modifications is the major research subject in this project. Development of novel experimental techniques for this purpose is an additional important research subject ^(242-²⁴⁴⁾. The Comprehensive Isolation of CpG Islands Methylated in Human Lung Cancer 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, a comprehensive isolation of CpG islands that are specifically methylated in human lung adenocarcinomas has been performed ⁽²⁴⁵⁾. Approximately 200 CpG islands that are methylated in tumor DNA has been isolated. These CpG islands include 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 the epigenetic dysregulation of gene expression by CpG island methylation. Following a comprehensive isolation of CpG islands that were methylated in human lung adenocarcinoma, it was shown 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. Many CpG islands at the HOXA and HOXD loci were methylated in human lung adenocarcinoma ⁽²⁴⁶⁾. The de novo methylation of these CpG islands was also observed in patient’s DNA from noncancerous portions of lung tissue. These results indicate the presence of specific chromosomal regions that are susceptible to de novo methylation. Heterogeneity in the Modification of Chromatin Components in Cancer Cells The structural alteration of chromatin has a key role in regulating gene expression. The alteration of chromatin is mediated by modification of its components. A detailed understanding of the relationship between these modifications, notably, methylation of the full-length CpG island, the association of methyl-CpG binding proteins (MBPs), and the acetylation and methylation of histones in gene silencing is vitally important. Currently however, the manner in which chromatin components, associated with a specific gene, are modified is poorly understood. The manner of the differential association between CpG methylation, MBPs, and histone modification in the entire CpG island of the human E-cadherin (CDH1) gene in cancer cells have been investigated ⁽²⁴⁷⁾. Of the cell lines with CDH1 transcriptional repression, the distribution of methyl-CpGs in the CpG island differed markedly. In a cell line with gene silencing, the promoter region was almost methylation-free. Chromatin immunoprecipitation analysis revealed that the acetylation status of histone H4 differed between cell lines. However, deacetylated histone H3 was associated with the CpG island in all silenced cell lines. Binding of MeCP2 was also detected in all silenced cell lines. Additional binding of the MBD1 protein was detected in a cell line in which the promoter region was poorly methylated and only histone H3 was deacetylated. Binding of MBD2 protein was detected in all other silenced cell lines. Histone H3 lysine 9 was methylated in all silenced cells, while histone H3 lysine 4 was methylated in some silenced cell lines. These results demonstrate that chromatin components associated with inactive CDH1 chromatin are heterogeneously modified and suggests the presence of multiple pathways for the formation of inactive chromatin.