Figure 4. NOR-1 regulates cIAP2 promoter activity through a NBRE site.

(a) cIAP2 proximal promoter sequence. Cis-acting regulatory elements and the putative NBRE are indicated. (b) Scheme showing the putative NBRE(−358/−351) site present in the cIAP2 promoter region cloned into the pGL3 reporter vector (pcIAP2-1808). The consensus NBRE site (WT-NBRE) is shown and mutated base pairs (m-NBRE) are indicated in bold. (c) Luciferase activity (normalized by Renilla) from cells co-transfected with a NOR-1 expression vector (pCMV5/NOR-1; black bars) or the corresponding empty plasmid (pCMV5; white bars) together with different pGL3/cIAP2 constructs. The activity of a construct mutated in the NBRE site (white triangle) is also shown (n = at least 6). *P < 0.0001 vs. cells co-transfected with pCMV5; ^#P < 0.0001 vs. cells co-transfected with pCMV5/NOR-1 and pcIAP2-1808. (d) Representative autoradiograms of EMSA performed with a cIAP2 probe containing the NBRE(−358/−351) site (NBRE) and nuclear protein extracts from VSMC transduced with lentiviral vectors to express NOR-1-FLAG (pNOR-1F) or EGFP (pGFP). The position of the complexes up-regulated by NOR-1 (I and II) is indicated. Competition assays with a molar excess of unlabelled probe (100-fold; Competitor) and supershift assays with a specific antibody against the FLAG sequence (anti-FLAG Ab) were performed. EMSA carried out with a mutated NBRE probe (m-NBRE) is also shown. SS: supershifted complex. (e) Representative image of an agarose gel electrophoresis corresponding to a ChIP assay showing the relative in vivo association of NOR-1 with the human cIAP2 promoter in VSMC that over-expressed NOR-1-FLAG (pNOR-1F), GFP (pGFP) or pLVX vector (pLVX). Sheared chromatin was immunoprecipitated with an anti-FLAG antibody (IP:FLAG) or a non-specific IgG (IP:IgG). The enrichment of NOR-1 was assessed by PCR using cIAP2 promoter specific primers. Equal input DNA and control IgG inmunoprecipitations are shown.