Cellular localization and colocalization of miR-223, YY1, and Dicer1. HL60 cells and primary APL blasts were treated or not (CTR) with 1μM RA for the indicated times and analyzed by immunofluorescence confocal microscopy. (A) Localization of miR-223–Mimic-Cy5 (Mimic-223) in HL60 cells and in APL blasts, β-tubulin marking cell cytoplasm, and the merge of the 2 channels. (B) Colocalization of Mimic-223, YY1 (AlexaFluor-488), and Dicer1 (AlexaFluor-555). Quadruple labeling (DAPI, YY1, Dicer1, Mimic-223) and double-channel visualization of untreated (CTR) or treated (RA) HL60 cells. Channel colors were palette assigned. A mask landmark (blue line) delineates the nuclear boundaries, and white arrows indicate areas of higher colocalization (yellow dots). (C) Acridine Orange-stained mitotic chromosomes, localization of 223-Mimic-Cy5 (Mimic-223), and the merge of the 2 channels. (D) ChIP assay performed on HL60 cells transiently transfected (+) or not (−) with Cy5-Mimic-223 and treated (+) or not (−) with 1μM RA for 48 hours. Chromatins were immunoprecipitated with an anti-Cy5 antibody. NFI-A regions 1, 2, and 3 were PCR amplified and visualized by ³²P-labeled oligonucleotides (supplemental Table 5) as described in supplemental Methods or by quantitative RT-PCR measuring Mimic-223 or Mimic-NC enrichment at NFI-A region 1 (bottom panel). Error bars represent SD of 3 independent evaluations. (E) Schematic model for the heterochromatic silencing of NFI-A gene by PcG-miR-223 complexes in RA-induced granulocytic differentiation. In undifferentiated HL60 cells (top panel), YY1 is present at low levels, with Suz12 and Ago1 at its binding sites on NFI-A promoter, and this correlates with an enrichment (↑) in active chromatin marks (acH3, H3K4me3), decreased (↓) repressive/inactive mark H3K27me3, demethylated CpGs (○), recruitment of RNAPol-II, and NFI-A transcriptional activation. On RA-induced granulocytic differentiation (bottom panel), miR-223 localizes in the nucleus and via the formation of a Dicer1/Ago1-YY1/Suz12 complex, miR-223 targets its complementary DNA sequences flanking YY1 binding sites on NFI-A promoter at a distance of approximately 1 nucleosome. The increased occupancy of the PcG-miR complexes at these sites correlated with a decrease in active H3K4me3 and an enrichment in repressive/inactive mark H3K27me3, CpG methylation (●), transcriptional silencing of NFI-A gene, and granulocytic differentiation of myeloid precursors. Numbers are the nucleotides relative to the start site of the NFI-A gene (+1).