Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans-Regulation of Gene Networks

Lesca M Holdt,Gerhard Schuler,Knut Krohn,Knut Finstermeier,Stephan Gielen,Hendrik Bergert,Anika Stahringer,Peter F Stadler,Daniel Teupser,Joachim Thiery,Wolfgang Wilfert,Ingo Bechmann,Steve Hoffmann,Gabor Gäbel,Frank Beutner,David Langenberger,Markus Scholz,Kristina Sass,Mark I Mccarthy

doi:10.1371/journal.pgen.1003588

Abstract

The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

Highlights

The chromosome 9p21 (Chr9p21) locus is the strongest genetic risk factor of atherosclerosis known today, yet, the responsible mechanisms still remain unclear
We show that increased antisense non-coding RNA in the INK4 locus (ANRIL) expression mediates atherosclerosis risk through transregulation of gene networks leading to pro-atherogenic cellular properties, such as increased proliferation and adhesion
Our work extends the function of Alu transposons to regulatory components of long non-coding RNA (ncRNA) with a central role in epigenetic transregulation

Summary

Introduction

The chromosome 9p21 (Chr9p21) locus is the strongest genetic risk factor of atherosclerosis known today, yet, the responsible mechanisms still remain unclear. The closest neighbouring genes are the cyclin-dependent kinase inhibitors CDKN2A and CDKN2B, which are located ,100 kb proximal of the Chr9p21 atherosclerosis risk region. While these genes are expressed in atherosclerotic lesions [7], the majority of studies in humans speak against a cis-regulation of CDKN2A and CDKN2B by Chr9p21 (reviewed by [8]). A correlation of ANRIL expression with atherosclerosis severity has been described [2,8]. Based on these clinical and experimental data, ANRIL must be considered as a prime functional candidate for modifying atherosclerosis susceptibility at the Chr9p21 locus

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