The miR-23a/24-2 and miR-210 promoters are repressed by PML-RARA. (A) ChIP experiments performed in NB4 cells. Chromatin was immunoprecipitated using the indicated antibodies and the enriched genomic fragments were PCR amplified using specific primers. 223-A and 223-B correspond to the 2 miR-223 promoters described in Fukao et al²⁰  and Fazi et al,¹⁷  respectively. The RARB promoter was used as a positive control. (B) qPCR analyses performed on DNA immunoprecipitated in panel A. The negative control corresponds to a sequence located 3.9 kb downstream the miR-23a/24-2 precursor. Fold enrichment over the negative control (no antibody) was calculated using the following formula: 2^{[(Ct input − Ct IP) − (Ct input − Ct no Ab)]}. (C) LUC assays performed in 293T cells transfected with a firefly luciferase reporter gene driven by the miR-23a/24-2 promoter together with the control empty pcDNA3 plasmid (black histograms) or the PML-RARA–expressing vector (gray histograms). A promoterless vector was used as a negative control (pGL3b). A miR-23a/24-2 promoter mutated in the predicted PML-RARA response element was also tested (mutated miR-23a/24-2 promoter). At 24 hours after transfection, 293T cells were treated or not with ATRA (1 μM) for 16 hours. Results are expressed as relative light units (RLUs), 1 representing the value obtained with the promoterless vector pGL3b in absence of PML-RARA and ATRA treatments.