Figure 2
DNA methylation functions in pre-mRNA splicing. (A) DNA methylation is involved in splicing. To detect the function of DNA methylation in splicing, RNAs isolated from 5azadC-treated and control (CTR) IMR90 cells were profiled using RNA-Seq. Similarly, RNAs isolated from HCT116-WT or HCT116-DKO cells were profiled using RNA-Seq. The significantly changed exons were identified using MISO from the RNA-Seq data. The table lists the numbers of the significantly downregulated and upregulated exons induced by DNA methylation inhibition. (B) Hypermethylated exons have a higher tendency to be downregulated than hypomethylated exons following inhibition of DNA methylation by 5azadC treatment in IMR90 cells. The degree of exon downregulation was calculated and compared between hypermethylated and hypomethylated included exons. High meDNA indicates ASE with > 70% CpG methylation and low meDNA indicates ASE with < 30% CpG methylation. P-value was calculated by one-sided KS test. (C) Inhibition of DNA methylation leads to skipping of exon 10 of the HAUS8 gene in the spliced product. UCSC browser display of indicated data (NCBI36/hg18 assembly) over coordinates chr19:17,021,565-17,029,029 for HAUS8 focusing on the last three RefSeq annotated exons. DNA methylation data from HCT116-WT and IMR90 cells were obtained from public sources and displayed accordingly15,46. Exon junctions from paired-end RNA-Seq data from 5azadC-treated IMR90 cells and HCT116-DKO cells and their respective control cells are displayed, where red indicates aberrant skipping events and black indicates normal “full-length” transcript. The number of tags representing each exon junction is shown to the left of the junctions. Exons are labeled according to RefSeq. meDNA: DNA methylation. (D) Comparison of DNA methylation in unperturbed (HCT116-WT) and demethylated (HCT116-DKO) cells in the HAUS8 exon 10 region. DNA methylation was determined by the bisulfite-sequencing of the HAUS8 exon 10 region. Each colored circle represents individual CpG cytosine methylation status within the analyzed region (black: methylated; white: unmethylated). Percentage of methylation was determined based on the number of methylated CpG cytosines divided by the total number of CpG cytosines within the analyzed sequences from all the PCR clones. P-value shown was calculated using one-sided Student's t-test.
