A Genome-wide Study Of Aberrant DNA Methylation And Histone Methylation Patterns In Melanoma

Part I Alterations of genome-wide DNA methylation patterns in melanomaObjectives To investigate the alterations of DNA methylation patterns in melanoma at a genome-wide level.Methods The genomic DNAs of melanoma cell lines SK-MEL-28, LOXIMVI and normal melanocytes HEMn, HEMa were extracted. Digesting the genomic DNA sequentially with SmaI and XmaI, a DNA library for Next-Generation Sequencing was built with the digestion product. By deep sequencing the DNA fragments in the library, the methylation information of each CpG site was acquired by calculating the ratio of SmaI-ends numbers to Xmal-ends numbers. This method is called Digital Restriction Enzyme Analysis of DNA Methylation (DREAM). Using normal melanocytes as controls, the alterations of DNA methylome in melanoma were analyzed.Results A large proportion of CpG sites locating out of CpG islands and outside promoter regions were abnormally hypomethylated in melanoma, while a proportion of sites within CpG islands were abnormally hypermethylated across the genome. The methylation levels of promoter CpG islands were reversely correlated with gene expression (Spearman r=-0.39,-0.57 for SK-MEL-28 and LOXIMVI, respectively). Genes with methylated promoter CpG islands in normal melanocytes are more likely to gain DNA hypermethylation in melanoma compared to genes with unmethylated promoter CpG islands (p<0.001).Conclusion Global DNA hypomethylation and widely CpG islands hypermethylation exist in the genome of melanoma. The methylation status of promoter CpG islands in normal cells determines the gene's inclination of gaining hypermethylation in melanoma.Part II Alterations of genome-wide histone methylation patterns in melanomaObjectives To analyze the abnormal methylation patterns of histone H3K4me3 and H3K27me3 in melanoma at a genome-wide level.Methods Global histone H3K4me3 and H3K27me3 patterns in melanoma cell lines SK-MEL-28, LOXIMVI and normal melanocytes HEMn, HEMa were analyzed by Chromatin Immunoprecipitation Sequencing (ChIP-seq).Results The distribution of H3K4me3 and H3K27me3 across the genome and their impact on gene expression were different in both melanoma cell lines and normal melanocytes. H3K4me3 was mainly enriched at promoter regions, and correlated with active gene expression (p<0.001). H3K27me3 tended to cover broader regions overlapping the gene areas, and correlated with gene repression (p<0.001). About 90% of the bivalent genes marked by both H3K4me3 and H3K27me3 in normal melanocytes lost either of their H3K4me3 or H3K27me3 marks in melanoma, and about 70% of the genes marked exclusively by H3K27me3 lost their H3K27me3 marks in melanoma. In contrast, about 70% of the genes marked exclusively by H3K4me3 maintain the same histone mark in melanoma, while only 30% or even less lost their H3K4me3 marks.Conclusion Significant changes of H3K4me3 and H3K27me3 patterns at a genome-wide level occur in melanoma.Part III Relationships between DNA methylation and histone methylation status in melanomaObjectives To analyze the relationships between DNA methylation and histone H3K4me3/H3K27me3 status in melanoma Methods The relationships between DNA methylation and histone H3K4me3 and H3K27me3 status in melanoma cell lines and normal melanocytes were analyzed at a genome-wide level using the data acquired by DREAM and ChIP-seq.Results In the same melanocyte or melanoma cell line, the enrichment of H3K4me3 was found higher at gene promoters with unmethylated CpG islands compared to at promoters with methylated CpG islands (p<0.001). Genes with H3K4me3 marks in normal melanocytes are less likely to be hypermethylated in melanoma compared with genes that are either marked with H3K27me3 or without H3K4me3 marks (p<0.001). About 17%-21% of hypermethylated genes in melanoma were marked with H3K4me3 marks in normal melanocytes.Conclusion Histone H3K4me3/H3K27me3 status are strongly correlated with DNA methylation and its alterations, which together participate in the carcinogenesis of melanoma.