This study provides the first comprehensive, detailed map of DNA methylation patterns in human mammary cell lines. Methylated DNA fragments were detected using a highly sensitive method involving enrichment by MeDIP, and high-throughput sequencing enabled the non-biased mapping of aberrant methylation sites across the genomes of BCC lines. We examined 10 different cell populations in total, including EMT-induced MCF7 cells, to investigate the association between DNA methylome alterations and changes in cellular morphological phenotypes. Our data sets covered almost the entire genome with sufficient depth to identify differentially methylated regions, thereby providing high resolution and reproducibility, and proved that MeDIP-seq is a cost-effective approach for comparative analyses of the mammalian DNA methylome. Although many researchers have sought to describe DNA methylome alterations in cancers, to our knowledge this is the first methylome study that effectively encompasses the entire genome and is not limited to specific sequences. This study has thus provided important new insights into the biological implications of DNA methylation.
Firstly, the global methylation map provides an indication of the intricate relationship between the methylome and transcriptome. Most cancer methylome studies have assumed that functionally important DNA methylation is restricted to promoters and that most DNA methylation changes in cancer occur in CGIs, and they ignore all other regions. In our analysis, although hypermethylation at predefined CGIs was a remarkable feature of the BCC lines, we also found that many non-CGI regions were broadly hypermethylated. Hypermethylation occurred not only at proximal promoters but also at exons and introns, including regions distal from the TSS. Since DNA methylation interrupts the binding of transcription factors to their response elements [2, 11], changes in methylation at distal regions may affect the expression of a gene. Since proximal promoters or CGI methylation are not always correlated with gene expression [13, 33], our comprehensive methylation maps will help us to better understand methylation-dependent transcriptional regulation.
Secondly, the comprehensive methylation maps help us to understand how methylation is maintained and how it is disrupted in BCC lines. We found that the number of reads was positively correlated with the number of CpGs within 100 kb segments and this relationship was especially clear in HMEC. Although HMEC showed relatively low methylation levels in gene-related CpG-rich regions (corresponding to less than 8% of whole-genome CpGs), overall, methylation of each CpG seemed to be maintained at a nearly constant level. The most prominent feature of aberrant methylation was the massive overall hypomethylation and simultaneous hypermethylation at CpG-rich regions. While these observations are consistent with previous reports , our results provide the most detailed map of aberrant methylation in BCC lines. We found that hypomethylation in BCC lines was biased towards CpG-poor regions, and analysis of genomic features indicated there was no preference for repeat sequences but included all gene-related regions equally. Furthermore, hypermethylations of CpG-rich gene-related regions was present in BCC lines although within extremely hypomethylated 100 kb segments. These results support the idea that there are at least two distinct maintenance mechanisms that produce the aberrant methylation patterns in CpG-poor regions and CpG-rich regions. One mechanism maintains CpGs at a constant methylation level, which is downregulated in BCC lines. Another mechanism maintains gene-related CpG-rich regions at a specific methylation level, which is generally upregulated in BCC lines.
Finally, the comparative analysis of methylation maps of MCF7 cells with or without EMT induction provided an increased understanding of how cells undergo DNA methylome alterations. Analysis of 100 kb segments revealed almost unchanged overall methylation levels throughout EMT, while hypomethylation was observed at many CGI sites. With respect to the two distinct mechanisms of methylome alteration described above, EMT had little effect on the maintenance of the overall methylation level, but had a considerable effect on cell-type specific methylation. On the other hand, EMT induction altered cell-type specific gene-related methylation such that hypomethylation was predominantly observed at CpG-rich regions. The idea that there are cell-type and gene specific mechanisms for regulation of methylation patterns within CpG-rich gene-related regions received further support from the following observations. The methylation status within CpG-rich regions were likely to be shared by BCC lines, and CpGs within CpG-rich repetitive sequences such as HSATII were extensively hypomethylated in BCC lines. Considering that the methylation status participates in determining the morphological phenotypes of the cells [7, 28, 29], our results suggested an interdependence between cell-type and gene specific regulation of DNA methylations and morphological changes during EMT.