Gfi1 loss of function directly deregulates HoxA9, Pbx1, and Meis1 in GMPs. (A) Quantitative real-time gene expression analysis of putative HoxA9-target genes c-Myb, CD34, Pim1, Flt3, Pu.1, Cxcr4, and Ebf1 in RNA from wild-type or Gfi1^−/− littermate Lin⁻ bone marrow cells. (B) Chromatin immunoprecipitation (ChIP) analysis with a Gfi1-specific monoclonal antibody (2.5D.17) or isotype control IgG (Con IgG) showing Gfi1 physically bound to conserved promoter elements in HoxA9, Pbx1, and Meis1 (Promoter), but not 3′-untranslated regions of these genes (3′UTR). Gfi1 binding was detected in control nontargeting shRNA-treated HL60 cells (HL60-NT shRNA) but not in Gfi1-specific shRNA-knock-down HL60 cells (HL60-Gfi1 shRNA). (C,D) Quantitative real-time gene expression analysis of Gfi1, Gfi1b, HoxA9, Meis1, Pbx1, and Pbx2 in sorted CMPs (C) and GMPs (D) from RosaCreER^T2+Gfi1^{fex4-5/fex4-5} Lin⁻ bone marrow cells with or without tamoxifen (OHT). (E) Quantitative real-time gene expression analysis of putative HoxA9-target genes c-Myb, CD34, Pim1, Flt3, Pu.1, Cxcr4, and Ebf1 in RNA from panel D. (F) Quantitative real-time gene expression analysis of HoxA locus gene expression in RNA from wild-type or Gfi1^−/− littermate isolated GMPs. (G) Quantitative real-time gene expression analysis of HoxA9 in CD34⁺ bone marrow cells from 3 healthy donors or a GFI1N382S patient. Error bars indicate SD. (H,I) Methylcellulose colony-forming assay with serial replating of HoxA7^−/− (H) or HoxA9^−/− (I) Lin⁻ bone marrow cells transduced with retrovirus expressing Gfi1 dominant-negative mutants (P2A or N382S) or an empty vector control. Error bars indicate SEM.