FIG. 6.

Mutations within the PIM disrupt cooperative DNA binding with Pbx proteins but not with Meis1. (A) Gel shift analysis of cooperativity between Pbx and Meis proteins and both wild-type EE-tagged Hoxa9 (see Materials and Methods) and EE-tagged mutants of Hoxa9 within the PIM or HD, using the DNA element TGATTTAT. N-terminal EE-tagged versions of Hoxa9 and Hoxa9 mutants were used only for gel shifts assaying proteins derived via in vitro coupled transcription-translation because they produced significantly larger quantities of protein than the untagged versions. Additions to each binding reaction are indicated above the lanes. Monomeric Hoxa9 proteins bound to DNA are denoted Hoxa9 proteins; the background band is designated Bkg; heterodimers of Hoxa9 with Pbx1 or VP16-Pbx1 comigrated and are collectively denoted Pbx:HoxA9; heterodimers of E2a-Pbx1 plus Hoxa9 are designated E2a-Pbx1:Hoxa9; Hoxa9-Meis1c heterodimers are designated Meis1c:HoxA9 at the right. (B) Hoxa9-WF derived from nuclear extracts of immortalized myeloid progenitors also fails to heterodimerize with endogenous Pbx proteins. Nuclear extract from myeloid progenitors immortalized by Hoxa9 (lanes 1 to 3), Hoxa9-WF (lanes 4 to 6), or Hoxa9 treated with G-CSF for 3 days (lanes 7 to 9) was subjected to gel shift analysis using the probe TGATTTAT. Monomeric binding by Hoxa9 is indicated at the left, showing three discrete monomeric Hoxa9 bands. Western blot analysis confirmed that the Hoxa9 proteins in nuclear extracts were degraded into three smaller discrete species (data not shown).