M33-MEIS1 heterodimerizes with PBX1 on or off DNA and nucleates the formation of a Polycomb group complex on MEIS1 target genes. (A) EMSA was performed with nuclear extracts from E86-Pbx1, E86-Pbx1+Meis1, and E86-Pbx1+M33-Meis1 viral producers (indicated at bottom) and incubated with a radiolabeled oligonucleotide containing a Meis1/Pbx1 consensus binding sequence.7 Each nuclear extract was incubated with probe and a combination of probe with molar excess of unlabeled (cold) probe, anti-HA antibody, or anti-PBX1 antibody. The MEIS1/M33-MEIS1:PBX1 nucleoprotein complexes are indicated. (B) Coimmunoprecipitation experiments were performed with nuclear extracts from E86-Pbx1, E86-Pbx1+Meis1, and E86-Pbx1+M33-Meis1 viral producers (indicated at top). PBX1 is specifically immunoprecipitated by an anti-HA antibody in Pbx1+Meis1 and Pbx1+M33-Meis1–overexpressing cells and recognized by an anti-PBX1 antibody. In the reciprocal experiment, MEIS1 and M33-MEIS1 are specifically immunoprecipitated by an anti-PBX1 antibody and recognized by an anti-HA antibody. (C-F) Schematic representation of Flt3 (C), Trib2 (D), and Hlf (E-F) showing the putative transcription start sites (arrows), the exons (black boxes), the 5′ untranslated region (empty box), and the position of the amplicons assayed in the ChIP analysis (striped box). The ND13+Meis1 and ND13+M33-Meis1–transduced BM cells were coimmunoprecipitated with the antibody indicated on the x-axis. The data represent the mean of fold enrichment relative to the immunoglobulin G (IgG) control samples ± SD of 3 independent experiments. AcH3 indicates acetylated histone H3; AcH4, acetylated H4; H3K27 tri-methyl, trimethylated Lys27 on H3.
