Identification of long noncoding RNAs associated to the EZH2 protein and their role in the epigenetic regulation in Pancreatic Cancer

Abstract

This project aims to investigate the contribution of long non-coding RNAs (lncRNAs) to epigenetic reprogramming and control of gene expression in Pancreatic Ductal Adenocarcinoma (PDAC). The hypothesis that motivates this study is that lncRNAs can interact with components of the epigenetic machinery, directing it to specific loci in order to modify chromatin activation/repression states, thus regulating gene expression to favour the establishment of PDAC malignant phenotypes. The focus of the study will be the identification and functional characterization of lncRNAs that interact with EZH2, the histone methyl transferase component of the Polycomb repressive complex 2 (PRC2). PRC2 participates in the epigenetic silencing of specific loci in key processes such as cell differentiation, cell cycle regulation and cancer. EZH2 can also act as a transcriptional activator through mechanisms independent of its catalytic activity and of PRC2. EZH2 is overexpressed in PDAC, but its mechanisms of action and target genes are not yet known. We will identify EZH2-associated lncRNAs through co-immunoprecipitation of the protein followed by high-throughput RNA sequencing (RIP-seq) in PDAC lines with different degrees of malignancy. We have recently identified a set of overexpressed lncRNAs in clinical cases of PDAC, as well as protein coding genes aberrantly expressed that have evidence of EZH2 occupancy in their promoter region. These results will help to prioritize lncRNAs and EZH2 target genes for detailed characterization. The candidate lncRNAs will be silenced and the expression and occupancy of EZH2 and the repressive H3K27me3 mark at the promoter region of target genes will be evaluated by RT-qPCR and ChIP-qPCR, respectively. The functional relevance for malignancy of the most promising lncRNAs will be assessed by phenotypic assays in pancreatic tumor cell models. (AU)