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 human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

Genome-Wide Profiling of Prognostic Alternative Splicing Signature in Colorectal Cancer

“…Present study found the epigenetic modifications of histones near the splicing sites affected the ratio of KRAS-4A vs. KRAS-4B by the differential AS patterns in colorectal cancer cell lines ( 27 ). In a very recent study, aberrant splicing regulation of the KRAS-4A vs. KRAS-4B transcript isoforms was associated with a higher level of KRAS signaling and poor prognosis, specifically in the microsatellite stable primary CRC ( 28 ).…”

ZNF518B gene up-regulation promotes dissemination of tumour cells and is governed by epigenetic mechanisms in colorectal cancer

“…The experimental results were compared with the output of the sequence-based prediction of positioning carried out using the NuPoP software tool 26 . Epigenetic modifications of histones were studied at nucleosomal level by Nuc-ChIP 10,25,27 . The following antibodies were used: anti-H3K9me3 (Abcam, Cambridge, UK, ab-8898); anti-H3K4me3 (Abcam, ab-8580); anti H3K9ac (Abcam, ab-4441); anti H3K27ac (Abcam, ab-4729).…”

The Intricate Interplay between Epigenetic Events, Alternative Splicing and Noncoding RNA Deregulation in Colorectal Cancer

“…Several studies have shown that histone marks decorate specific exons to regulate RNA Pol II elongation and thus influence co-transcriptional splicing [108,109,110]. For example, Poly (ADP) ribose polymerase (PARP1) marks histones and thereby changes nucleosome deposition at specific exon-intron boundaries, which in turn affects RNA Pol II movement and finally alters AS decisions in a context-specific manner [110].…”

“…For example, Poly (ADP) ribose polymerase (PARP1) marks histones and thereby changes nucleosome deposition at specific exon-intron boundaries, which in turn affects RNA Pol II movement and finally alters AS decisions in a context-specific manner [110]. Riffo-Campos et al showed that low nucleosome occupancy, due to differential histone marks at exon 4A of KRAS , resulted in an accelerated RNA Pol II elongation rate and subsequent lower abundance of isoform 4A in the CRC cell lines HCT116 and SW48 [109]. Yuan et al demonstrated in a CRC mouse model that mutation of the histone methyltransferase SETD2 slows down transcription elongation and thereby facilitates the removal of intron 2 of dishevelled segment polarity protein 2 (DVL2) pre-mRNA; thereby augmenting Wnt/β-catenin signalling and tumorigenesis.…”