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

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.

Author Summary

Chromosome 9p21 is the strongest genetic factor for coronary artery disease and encodes the long non-coding RNA (ncRNA) ANRIL. Here, we show that increased ANRIL expression mediates atherosclerosis risk through trans-regulation of gene networks leading to pro-atherogenic cellular properties, such as increased proliferation and adhesion. ANRIL may act as a scaffold, guiding effector-proteins to chromatin. These functions depend on an Alu motif present in ANRIL RNA and mirrored several thousand-fold in the genome. Alu elements are a family of primate-specific short interspersed repeat elements (SINEs) and have been linked with genetic disease. Current models propose that either exonisation of Alu elements or changes of cis-regulation of adjacent genes are the underlying disease mechanisms. Our work extends the function of Alu transposons to regulatory components of long ncRNAs with a central role in epigenetic trans-regulation. Furthermore, it implies a pivotal role for Alu elements in genetically determined vascular disease and describes a plausible molecular mechanism for a pro-atherogenic function of ANRIL at chromosome 9p21.