Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene 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. 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. Here we provide in vivo evidence in cancer cells of the differential association between CpG methylation, MBPs, and histone modification in the entire CpG island of the human E-cadherin (CDH1) gene. 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 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 is heterogeneously modified and suggests the presence of multiple pathways for the formation of inactive chromatin.