Fig. 7

Identification of functional MafB binding sites in Krox20 enhancer B. (A) Alignment of zebrafish, Xenopus, chick and mouse Krox20 element B nucleotide sequences showing the two conserved putative MafB sites MafB-1 and MafB-2 (red boxes). A sequence of lower similarity to the MafB consensus binding site, adjacent to MafB-1 and in the reverse orientation, is also indicated (dashed red box). A vHnf1 binding site is indicated by the green box. The oligonucleotides used for gel retardation (wtM1 and wtM2) are indicated beneath. (B) Alignment of MafB-1 and MafB-2 with the consensus MafB recognition element (MARE) half-site (W=A or T). The mutations introduced into the MafB-1 and MafB-2 sites are indicated in red. (C) Gel retardation analyses were performed with the indicated bacterial protein extracts (c, control without MafB protein) and oligonucleotides carrying the chick versions of the MafB-1 and MafB-2 sites, either wild-type (wtM1, wtM2) or mutated (mutM1, mutM2). FP, free probe. The bracket indicates MafB-probe shift complexes, which are abolished or largely abolished by mutation of the MafB-2 and MafB-1 sites, respectively. (D-K) Chick embryos were analysed by X-gal staining after electroporation with constructs containing wild-type (D,H-J) or mutant (E-G,K) versions of chick element B driving a β-globin promoter-lacZ reporter. Embryos were co-electroporated with MafB alone (H), vHnf1 alone (I), or both (J,K). In all cases, a Cherry-expressing vector was co-electroporated to monitor electroporation efficiency; Cherry visualisation (red) was carried out following X-gal staining and is shown to the left of each image. Note that strong X-gal staining quenches Cherry fluorescence. ov, otic vesicle.