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

<div><p>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) <i>antisense non-coding RNA in the INK4 locus</i> (<i>ANRIL</i>). <i>ANRIL</i> expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which <i>ANRIL</i> regulates target-genes <i>in trans</i>, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, <i>trans</i>-regulation was dependent on Alu motifs, which marked the promoters of <i>ANRIL</i> target genes and were mirrored in <i>ANRIL</i> RNA transcripts. <i>ANRIL</i> 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 <i>ANRIL</i> over-expression. The functional relevance of Alu motifs in <i>ANRIL</i> was confirmed by deletion and mutagenesis, reversing <i>trans</i>-regulation and atherogenic cell functions. <i>ANRIL</i>-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 <i>ANRIL</i> at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.</p></div

Image_1_Novel Mutations in the Asparagine Synthetase Gene (ASNS) Associated With Microcephaly.PDF

<p>Microcephaly is a devastating condition defined by a small head and small brain compared to the age- and sex-matched population. Mutations in a number of different genes causative for microcephaly have been identified, e.g., MCPH1, WDR62, and ASPM. Recently, mutations in the gene encoding the enzyme asparagine synthetase (ASNS) were associated to microcephaly and so far 24 different mutations in ASNS causing microcephaly have been described. In a family with two affected girls, we identified novel compound heterozygous variants in ASNS (c.1165G > C, p.E389Q and c.601delA, p.M201Wfs^∗28). The first mutation (E389Q) is a missense mutation resulting in the replacement of a glutamate residue evolutionary conserved from Escherichia coli to Homo sapiens by glutamine. Protein modeling based on the known crystal structure of ASNS of E. coli predicted a destabilization of the protein by E389Q. The second mutation (p.M201Wfs^∗28) results in a premature stop codon after amino acid 227, thereby truncating more than half of the protein. The novel variants expand the growing list of microcephaly causing mutations in ASNS.</p

Validation of <i>ANRIL</i>-associated cellular effects in primary cells and schematic of molecular scaffolding by <i>ANRIL</i>.

<p>(A) PBMC from carriers of the Chr9p21 CAD-risk allele defined by rs10757274, rs2383206, rs2383297, and rs10757278 (n = 8) showed increased adhesion (<i>P</i> = 0.001) and (B) decreased apoptosis (<i>P</i> = 0.008) compared to cells of carriers of the protective allele (n = 8). (C) Schematic of molecular scaffolding by <i>ANRIL</i> mediated through potential chromatin-RNA interaction by Alu motifs.</p

Annotated <i>ANRIL</i> transcripts in the Chr9p21 region, transcript structure and association of <i>ANRIL</i> isoforms with Chr9p21 genotype.

<p>(A) Chr9p21 haplotype structure (HapMap CEU, r²) and core atherosclerosis region (between rs12555547 and rs1333050). (B) Exons of initially discovered <i>ANRIL</i> transcripts and their relative position in the Chr9p21 region. *A full list of currently annotated <i>ANRIL</i> isoforms is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588.s001" target="_blank">Figure S1B</a>. (C) <i>ANRIL</i> transcripts identified by RACE and PCR amplification (L1-L17). 4 major isoform groups with 4 distinct transcriptions ends were identified. Frequency of exon occurrence/isoform group is color-coded and given in %. <i>ANRIL1-4</i> denote highly expressed consensus transcripts harbouring exons found in >50% transcripts of the respective isoform group (red). Dotted lines indicate positions of transcript-specific qRT-PCR assays. (D) Study design of <i>ANRIL</i> expression and association with Chr9p21. (E) Association of <i>ANRIL</i> isoforms with Chr9p21 in human PBMC, whole blood and vascular tissue (effect, % change/risk allele defined by rs10757274, rs2383206, rs2383297, and rs10757278).</p

<i>ANRIL</i> regulates gene expression in t<i>rans</i> and affects cell adhesion, metabolic activity, proliferation, and apoptosis.

<p>(A) Consensus transcripts of 4 <i>ANRIL</i> isoform groups identified by RACE and PCR (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588.s001" target="_blank">Figure S1</a>). Dotted lines indicate positions of transcript-specific qRT-PCR assays. (B) RT-PCR confirmation of <i>ANRIL</i> over-expression in stable ANRIL1-4 cell lines. 3–4 cell lines per <i>ANRIL</i> isoform were established, no effect on house-keeping gene expression was found (<i>beta-actin</i> (<i>BA</i>); <i>glyceraldehyde-3-phosphate dehydrogenase</i> (<i>GAPDH</i>)). (C) Heatmap of <i>ANRIL trans</i>-regulated transcripts corresponding to panel B. Transcripts with average down- (<0.5-fold, red) and up- (>2-fold, green) regulation relative to vector control are shown. (D–F) Adhesion of <i>ANRIL</i> over-expressing cell lines to PBS-, Matrigel-, and collagen-coated wells (<i>P</i><0.01 for comparison of ANRIL1, 2, 3, 4 vs. vector control). (G–I) Cell proliferation and metabolic activity determined by (G) absolute cell numbers, (*/^#<i>P</i><0.05 for ANRIL2 and ANRIL4 vs. vector control), (H) glucose utilisation, and (I) viability assay. (J–L) Apoptosis determined by (J) AnnexinV-positive cells, (K) caspase activity, and (L) caspase-3 staining. (H–K) <i>P</i><0.05 for ANRIL2 and ANRIL4 vs. vector control. (M–O) Reversal of effects by RNAi against <i>ANRIL</i> (*<i>P</i><0.05; SCR- scrambled control). (D–K) At least triplicate measurements per pool of 3–4 biological replicates were performed. For details on experimental setup and <i>P</i>-values please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588.s012" target="_blank">Table S3</a>. (M–O) n = 3/group. Validation of siRNA knock-down is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588.s006" target="_blank">Figure S6</a>. Error bars indicate s.e.m.</p

Results of associations between gene-expressions and metabolites.

<p>Table displays significant associations of eQTL genes and metabolites for validated loci. Genes and metabolites are ordered according to strength of association. Statistics of strongest associations are also presented.</p><p>¹Gene with strongest association is presented in <b>bold</b></p><p>²Metabolite with strongest association is presented in <b>bold</b></p><p>³A q-value<5% was considered as significant, i.e. FDR is controlled at 5%.</p><p>Results of associations between gene-expressions and metabolites.</p

GWAS results for amino acids (a) and acylcarnitines (b) in whole blood.

<p>Manhattan plots of the genome-wide association analysis for metabolic phenotypes in 2,107 individuals of the LIFE-Heart cohort. Results are presented separately for 36 acylcarnitines (including free and total carnitine) and 26 amino acids. Results for metabolite ratios are omitted. The horizontal line represents a p-value = 1.0x10^-7, which was the cutoff used for inclusion of identified associations in the replication state.</p

Identification of Alu motifs in promoters of <i>ANRIL trans</i>-regulated genes and <i>ANRIL</i> RNA and their spatial relation to PcG protein binding.

<p>(A) DNA motif in promoters (5 kb) of <i>trans</i>-regulated genes representing an Alu-DEIN repeat <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588-Deininger1" target="_blank">[33]</a>. (B) Number of Alu motifs in promoters of <i>trans</i>- and not regulated transcripts (n per 5 kb). (C) ChIP-seq enrichment of SUZ12 and CBX7 binding distal to Alu motif, demonstrating a specific spatial relation of motif occurrence to PcG protein binding. RPM- reads per million mapped reads, CTR- random DNA control sequence. (D) Motif-associated SUZ12 signal peaks in an independent data set from BGO3 cells. The actual signal peak only becomes apparent, if a multiple matching policy is adopted. (E) Using a strict unique-matches only policy, a substantial signal reduction is seen downstream of the motif. Please note differences in y-axis scaling in (D, E). (F) Secondary RNA structure prediction for ANRIL2 using the Vienna RNA package <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588-Tacker1" target="_blank">[58]</a>. Within the minimum free energy structure, the Alu-DEIN motif is located in a stem-loop structure (arrow). (G) RIP demonstrating <i>ANRIL</i> binding to histone H3 (H3) and trimethylated lysine 27 of histone 3 (H3K27me3) in ANRIL2 (blue) and ANRIL4 (red) cells. <i>U1</i> was used as negative control. mIgG/rIgG- mouse/rabbit IgG control. Error bars indicate s.e.m.</p

<i>ANRIL</i> binds to PRC1 and 2 proteins and recruits CBX7 and SUZ12 to promoters of target genes.

<p>(A, B) RNA immunoprecipitation (RIP) followed by qRT-PCR demonstrating <i>ANRIL</i> binding to PRC but not to CoREST/REST proteins in (A) ANRIL2 and (B) ANRIL4 cells. Copies of ANRIL relative to input control are given in (A) blue and (B) red, nuclear ncRNA <i>U1</i> (white) was used as negative control. rIgG/mIgG/gIgG- rabbit/mouse/goat IgG controls. Error bars indicate s.e.m. (C,D) SUZ12 binding in promoters of <i>ANRIL</i> up-(green), down-(red), and not (black) regulated genes in (C) vector control cell line and (D) in BGO3 cells (GSM602674). TSS- transcription start site. (E, F) Effect of <i>ANRIL</i> over-expression on (E) SUZ12 and (F) CBX7 binding in promoters of up-regulated genes (vector control- dotted line vs. ANRIL2- straight line). (G) Reversal of <i>ANRIL trans</i>-regulation by RNAi against SUZ12 and CBX7 in ANRIL2 cells. SCR- scrambled siRNA control.</p

Pivotal role for Alu motif in <i>ANRIL</i> RNA function.

<p>(A) Cell lines over-expressing variants of <i>ANRIL2</i> (ANRIL2a, 2b, 2c) and <i>ANRIL4</i> (ANRIL4a, 4b) devoid of Alu motif sequences highlighted by boxes. Validation of over-expression using qRT-PCR assays. (B) Reversal of up-regulation (<i>TSC22D3</i>) and (C) down-regulation (<i>COL3A1</i>) of <i>ANRIL</i> target-gene mRNA expression in ANRIL2a-2c and ANRIL4a,4b compared to ANRIL2 and ANRIL4. Reversal of (D) cell adhesion, (E) apoptosis, and (F) proliferation in cell lines over-expressing mutant <i>ANRIL</i> isoforms. (G) Stable cell lines containing mutated forms of the Alu motif in <i>ANRIL2</i>. Positions of nucleotide exchanges (0, 25%, 33%, and 100%) in the 48 base-pair Alu motif are indicated in red. (H,I) Reversal of <i>trans</i>-regulation in mutant cell lines compared to ANRIL2. (J) Cell adhesion, (K) apoptosis, and (L) proliferation in mutant cell lines. (B,C,H,I,F,L) 3–4 biological replicates/isoform.(D,E,J,K) quadruplicate measurements per pool of 2–3 biological replicates. Error bars indicate s.e.m.</p