Fig. 6. Consensus bipartite HOX/PBC sites in the enhancer and their properties. (A) List of characterized HOX/PBC sites and Meis/Prep sites found in target genes aligned with those detected in Hoxa3. Mutated sequences used in binding and transgenic assays are indicated. (B,C) Reporter expression in 10.0 dpc mouse embryos carrying a construct with five copies of the HOX/PBC-A site linked to lacZ. Note strong expression in r5/r6; arrowhead marks posterior neural expression. (D,E) Electrophoretic mobility shift assays where a labeled double-stranded oligonucleotide containing the Hoxb2 HOX/PBC site and its associated Prep/Meis-binding site (B2-PP2)(Ferretti et al., 2000) has been mixed with the combinations of Pbx1a, Prep1 and Hoxb1 proteins (noted above the panels) in the absence or presence of varying amounts of cold competitor oligonucleotides spanning the HOX/PBC-A site (A3-PP2; E) or the HOX/PBC-B site (A3-PHP1; D). MUTA and MUTB are mutant forms of the competitors carrying the changes noted in A. Arrows at the sides indicate shifted complexes interacted with dimeric and trimeric combinations. (F) Gel shift assay where a labeled double-stranded oligonucleotide containing the Hoxa3 HOX/PBC-B site (A3-PH1; Fig. 3) has been mixed with the combinations of Pbx1a, Hoxb3, Hoxa3 or Hoxd3 proteins (noted above the panels) in the absence or presence of a 100 times excess of cold competitor oligonucleotides containing the wild-type (A3-PH1) or mutated form of the HOX/PBC-B site (MUTB). The addition of anti-Pbxa antibodies (
-Pbxa) inhibits complex formation.
