Deregulation of Hoxa9 expression and epigenetic modification in myeloid cells from Gfi1^36N/36N knock-in mice. (A) Hoxa9 mRNA expression was determined in GMPs by RT-PCR. One representative experiment (with triplicates for each experiment) from 2 independent experiments is shown. (B) Schematic representation of the Hoxa9 locus. Numbers indicate the position of the primer pairs used for ChIP-PCR. (C) ChIP-PCR with sorted Lin⁻, c-kit⁺, Sca1⁻ cells from the indicated mouse strains using an α-Gfi1 antibody. Shown is the representative result of 2 independent experiments, each done in triplicate (for location of primers see panel B). (D) ChIP-PCR with sorted Lin⁻, c-kit⁺, Sca1⁻ cells from the indicated mouse strains using an α-H3K4 dimethyl-antibody (for locations of primers see panel B). Enrichment for Gfi136S is set at 1. (E) ChIP-PCR with sorted Lin⁻, c-kit⁺, Sca1⁻ cells from the indicated mouse strains using an α-H3K9dimethyl-antibody (for locations of primers see panel B; for enrichment see panel D). (F) ChIP-PCR with sorted Lin⁻, c-kit⁺, Sca1⁻ cells from the indicated mouse strains using an α-H3K9acetyl antibody (for locations of primers see panels B and D). (G) Immunoprecipitations (IP) using thymocyte extracts from the indicated mouse strains with an α-Gfi1 antibody. As control, an α-actin antibody was used. (H) ChIP-PCR with thymocytes using an anti-Gfi1 antibody (from left to right: Gfi1^+/+, Gfi1^36S/36S, Gfi1^36N/36N). Relative enrichment was determined by amplification with previously described primers (see “ChIP”). (I) Hoxa9 mRNA expression level was determined in blast cells of 4 GFI1^36S/36S and 4 GFI1^36N/36S AML patients (P ≤ .10).