Mysm1 mediates localization of PU.1 to the Flt3 locus, but not to the GM-CSF-α receptor and M-CSF receptor loci. (A) ChIP analyses of PU.1 recruitment in WT or Mysm1^−/− BM Lin⁻ cells. The precipitated DNA was analyzed by qPCR via 2 sets of primers amplifying the Flt3 promoter region (promoter 1 and promoter 2) or the first exon (exon1) and normalized with IgG control. (B) Sequential 2-step ChIP analyses of Mysm1 and PU.1 recruitment to the Flt3 promoter in WT BM Lin⁻ progenitors. Chromatin was immunoprecipitated with anti-Mysm1 or IgG for the first ChIP. The pulled-down chromatin was eluted and reimmunoprecipitated with anti-Pu.1 or IgG for the second ChIP. The released DNA from the first and second ChIP were subjected to qPCR analysis using primers encompassing Flt3 promoter. Relative binding was defined by determining the ratio of immunoprecipitated DNA to that in the input sample. The ratios of the control IgG-immunoprecipitated DNA to that in the input samples were set as 1.0. (C) Coimmunoprecipitation assay of Mysm1 and PU1. pCMV-Pu.1 plasmid was cotransfected with either pCMV-Flag-Mysm1 or pCMV-Flag expression plasmid into HEK293T cells and cell lysates were incubated with anti-Flag antibody and immunoprecipitated proteins were analyzed by anti-PU.1 antibody via western blot analysis. (D) ChIP analysis of Mysm1 and PU.1 recruitment to GM-CSF-α receptor or M-CSF receptor gene loci in WT Lin⁻ cells. The signal was normalized to IgG control. (E) ChIP analysis of PU.1 recruitment to the promoter loci of GM-CSF-α receptor and M-CSF receptor. Lin⁻ cells were isolated from Mysm1^−/− BM and chromatin was immunoprecipitated with anti-PU.1 or IgG. The pulled-down DNA was eluted and amplified by qPCR via primers spanning the promoter or exon regions of GM-CSF-α receptor and M−CSF receptor. The signal was normalized to IgG control. (F) The qPCR analyzing messenger RNA of GM-CSF-α receptor (GM-CSFR) and M-CSF receptor (M-CSFR) in WT and Mysm12/2 Lin2 cells. *P < .05; **P < .01; ***P < .001.