Fig. 2. Trans-acting factors from E13.5 mouse embryonic brain bind to the I12b enhancer. (A) DNase I footprinting analysis of two overlapping DNA fragments of the I12b enhancer (nt 1-292 and 259-437) using a nuclear protein extract from E13.5 mouse forebrain. Six regions of the enhancer (FP1 to FP6) are bound by nuclear proteins. Protected areas (footprints) are represented by thick lines and hypersensitive sites by arrowheads. Numbers to the left of each panel indicate relative positions on the enhancer. (B) Protein-DNA interactions mapped (using GA Sequence in A) on the mouse I12b enhancer sequence. The protected areas identified by DNase I footprinting (FP1-FP6) are boxed. The two fragments used for the footprinting analysis are indicated by colored arrows (red arrows, nt 1 to 292; blue arrows, nt 259-437). Sequences of the human (H) and zebrafish (Z) I12b sequences are compared with mouse (M). Protein-DNA interactions are mostly located in regions with a high degree of sequence similarity. (C) Reporter constructs containing mutations (red) of the FP sites in the I12b enhancer.