RNA-dependent recruitment of YY1 and Dicer1 at NFI-A regulatory region 1. (A) RNAse A or RNAse H (10 μg/mL) was added for 3 hours to nuclei isolated from HL60 cells treated with RA 1μM for 72 or 96 hours before chromatin cross-linking. ChIP assay was performed using anti-Dicer1, -YY1 antibodies and analyzed by quantitative RT-PCR using the indicated primers. Data are mean ± SD of 2 independent evaluations. (B) RNA immunoprecipitation assay performed on HL60 cells untreated (CTR) and treated 1μM RA for the indicated time points to test endogenous miR-223, Let-7a3, and miR-126 binding to YY1, Dicer1, and Ago1 proteins. Cell lysates were immunoprecipitated with antibodies anti-YY1, -Dicer1, and -Ago1. Rabbit IgGs were used as specificity controls (not shown). Immunoprecipitated miRs were detected by quantitative RT-PCR as described in “RNA immunoprecipitation.” Error bars represent SD of 3 independent evaluations. (C) Nucleotide sequence of the −1.4-kb regulatory region surrounding and including NFI-A region 1. The transcription start site (+1) is marked with a rightward-pointing arrow. Red represents the YY1 binding sites; green, the sequences complementary to that of miR-223; and blue, GAGAG recurrent motifs. A black box represents the CpGs cluster region. CG dinucleotides evaluated by bisulphite sequencing assay are marked in italic and bold. The arrows indicate the position of the primers used to amplify the NFI-A region 1. (D) Genomic site conservation and relative percentage of the miR-223 target sequences (miR-223 site 1 and site 2) in the NFI-A gene promoter. Green represents highly conserved nucleotides.