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

Figure 3

ANRIL binds to PRC1 and 2 proteins and recruits CBX7 and SUZ12 to promoters of target genes.

(A, B) RNA immunoprecipitation (RIP) followed by qRT-PCR demonstrating ANRIL 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 U1 (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 ANRIL 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 ANRIL 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 ANRIL trans-regulation by RNAi against SUZ12 and CBX7 in ANRIL2 cells. SCR- scrambled siRNA control.

Figure 3