Fig. 6. A conserved Hoxb4-binding site is required for Rarb distal enhancer activity in chick embryos. (A) Map of the 5' end of the Rarb gene. Transcripts for the RARß1 and RARß3 isoforms initiate at the P1 promoter and those of the RARß2 and RARß4 isoforms at the P2 promoter. The positions of the RARE (blue oval; proximal enhancer) and the Hox/Pbx (HP) element (green oval, distal enhancer) are shown. White and grey boxes indicate transcribed non-coding and coding sequences, respectively. The positions of the 2.3 kb distal enhancer and the 3.8 kb proximal enhancer are also shown. Exon E1', which is equivalent to E4 in previous publications, is relabelled here to emphasize that it is the first exon of Rarb2/4 transcripts and not contiguous with Rarb1/3 transcripts. (B) Alignment of the Hox/Pbx (HP) element and flanking sequences in five mammalian species. The HP site is boxed in green and indicated below is the region multimerized for the 3xHP construct used in F. (C,D) Electrophoretic mobility shift assays showing Hoxb4 binding to the HP site. Labelled oligonucleotides containing the HS1+HS2 site (Gould et al., 1997) or the HP site (from Rarb) were incubated with increasing amounts of Hoxb4 protein (C) or with a constant amount of Hoxb4 protein in combination with Hoxb4 antibody (Hoxb4) or unlabelled competitor oligonucleotides as indicated (D). (E-G) Dorsal views of chick embryos electroporated with lacZ reporter constructs containing the mouse 2.3 kb distal enhancer (E), 3xHP oligonucleotide (F) or 2.3 kb distal enhancer with mutated HP site (G). lacZ expression with an r6/r7 boundary can be observed on the electroporated (right) side of the neural tube, except when the distal enhancer containing the mutated HP site (G) was used. The positions of the otic vesicle (OV) and the r6/7 boundary (arrowhead) are shown. Constructs are diagrammed below each panel, the circle indicating intact HP site (green fill) or HP MUT site (cross).