Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines
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Ángela L. Riffo-Campos1,2, Francisco Gimeno-Valiente1, Fernanda M. Rodríguez1,3, Andrés Cervantes1,4,5, Gerardo López-Rodas1,6, Luis Franco1,6 and Josefa Castillo1,5
1Institute of Health Research INCLIVA, Valencia, Spain
2Present Address: Centro De Excelencia de Modelación Y Computación Científica, Universidad De La Frontera, Temuco, Chile
3Instituto De Ciencias Veterinarias Del Litoral (ICIVET Litoral), Universidad Nacional Del Litoral (UNL)/Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Santa Fe, Argentina
4Department of Medicine, Universitat De València, Valencia, Spain
5Centro De Investigación Biomédica En Red En Cáncer (CIBERONC), Madrid, Spain
6Department of Biochemistry and Molecular Biology, Universitat De València, Valencia, Spain
Luis Franco, email: [email protected]
Keywords: epigenetics; chromatin structure; alternative splicing; colorectal cancer; KRAS isoforms
Received: November 03, 2017 Accepted: March 17, 2018 Published: April 17, 2018
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.
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