Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines

Abstract

Mutation-driven activation of KRAS is crucial to cancer development. The humangene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Theirdifferent properties and oncogenic potential have been studied, but the mechanismsdeciding the ratio 4A/4B are not known. To address this issue, the expression of thefour KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116and SW48 were further selected because they present the highest difference in theratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure wasanalysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at thetwo flanking exons. The low nucleosome occupancy at exon 4A in both cell lines mayresult in a fast transcriptional rate, which would explain the general lower abundanceof isoform 4A, also found in cells and tissues by other authors, but due to its similaritybetween both cell lines, chromatin structure does not influence alternative splicing.DNA methylation downstream exon 4A significantly differs in HCT116 and SW48cells, but the CCCTC-binding factor, which affects the processivity of RNA polymeraseand the alternative splicing, does not bind the differentially methylated sequences.Quantitative epigenetic analysis at mononucleosomal level revealed significantdifferences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac,H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes altersthe ratio 4A/4B. It can be concluded that the epigenetic modification of histones hasan influence on the selection of isoforms 4A and 4B.