Abstract 3145: Non-coding promoter-associated RNAs (PARs) orchestrate the epigenetic control of CDH1 transcription in cancer cells

Abstract

Abstract DNA in eukaryotic cells is wrapped around histones to form chromatin fibers. Chromatin can assume different conformations that dictate its biological activity. The equilibrium between distinct chromatin states is achieved via a cascade of epigenetic events involving DNA and histone modifications. However, the underlying mechanisms determining the timing and selectivity of these epigenetic events are not fully understood. Alterations in epigenetic patterns are common in cancer leading to the aberrant silencing of tumor suppressor genes. The CDH1 gene encodes the epithelial cell adhesion molecule E-cadherin and is frequently silenced in epithelial tumors. CDH1 silencing is associated with cell transformation, tumor invasiveness and metastatic dissemination along with epithelial-mesenchymal transition and acquisition of stem cell properties. Recently, non-protein coding RNAs (ncRNAs) and components of the RNA interference machinery have been proposed as important elements in epigenetic mechanisms that might assist in targeting epigenetic effectors to specific genomic loci. In this study, we investigated the role of non-coding promoter-associated RNA (PARs) in the transcriptional regulation of the CDH1 gene. PARs with both sense and antisense orientation relative to the protein-coding RNA were found in the CDH1 gene using strand-specific RT-PCR and 5’RACE. Sense and antisense PARs were positively and negatively correlated, respectively, to the level of CDH1 RNA in different cell lines, suggesting a functional link between coding and non-coding RNAs. RNA immunoprecipitation showed binding of the Argonaute protein Ago1 to the sense PAR in PC3 cells, in which the CDH1 gene was silenced. Knock-down of Ago1 led to re-expression of CDH1 along with increased histone H3 acetylation and decreased histone H3K9 methylation in the CDH1 promoter. Similar changes in the histone code and re-expression of CDH1 were induced by knocking down the H3K9 histone methyltransferase SUV39H1 and HDAC1. Moreover, Ago1 knock-down reduced binding of both SUV39H1 and HDAC1 to the CDH1 promoter, indicating a critical role of Ago1 in establishing their association with the promoter. Consistent with a role of the PARs in CDH1 transcriptional control, targeting the sense PAR with small interfering RNAs induced chromatin remodeling and CDH1 re-expression, supporting the hypothesis that PARs can direct epigenetic effectors to gene promoters through their interaction with Ago1. These findings suggest the existence of endogenous ncRNA-based mechanisms of transcriptional control that cooperate with and complement DNA and protein based epigenetic pathways in human cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3145.