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. 2016 Apr 28;37(7):656–668. doi: 10.1093/carcin/bgw048

Figure 2.

Figure 2.

RSV changes the landscape of DNA methylation in breast cancer cells and increases DNA methylation in genes that are hypomethylated in breast cancer cells. (A,B) Pie charts of differentially methylated CpG sites with the difference of at least 0.05 between RSV-treated and control MCF10CA1h (A) and MCF10CA1a (B) breast cancer cells (P ˂ 0.05, limma t-test). The landscape of DNA methylation was delineated using Illumina 450K microarray platform in breast cancer cells treated with RSV at 15 µM for 9 days (n = 3 per group). Diff. refers to differential methylation (delta beta, RSV minus control). (C,D) Chromosomal views of differentially methylated CpG sites. Each CpG site with significant difference of at least 0.05 between RSV and control cells was plotted into a bar track of a chromosomal view using IGV visualization tool. Each bar corresponds to a single, differentially methylated CpG site, with blue indicating hypomethylation and red indicating hypermethylation in RSV versus control (RSV minus Ctrl). (E) Venn diagram showing overlap between the hypermethylated genes in lowly invasive MCF10CA1h and highly invasive MCF10CA1a breast cancer cells in response to RSV. (F,G) Basal levels of methylation of ‘hypermethylated RSV targets’ as determined by the genome-wide microarray data in untreated MCF10A (normal), MCF10CA1h (cancer lowly invasive) and MCF10CA1a (cancer highly invasive) cells (F), as well as in MCF10CA1h and MCF10CA1a breast cancer cells exposed to 15 µM RSV for 9 days (G). The basal level of methylation of these genes in breast cancer cells is compared with the methylation levels in MCF10A cells. (H,I) Functional analyses using GO, KEGG and DAVID knowledgebase indicate biological functions (H) and pathways (I) associated with genes corresponding to CpG sites hypermethylated in response to RSV in both MCF10CA1h and MCF10CA1a. (J) Transcription factors and their binding motifs enriched for RSV hypermethylated genes in breast cancer. Putative transcription factor binding sites were predicted using TransFac. Percentage of RSV hypermethylated genes containing the motif is indicated.