Fig. 4. Gel-shift and footprint assays define two Brn3a-binding sites in the TrkA minimal enhancer. (A) Illustration of six, overlapping, 100 bp fragments covering TrkA minimal enhancer and (B) gel-shift using a GST-Brn3a POU-domain fusion protein indicating that only the first and sixth fragments can bind Brn3a. (C,D) DNAse I footprint assays to define the Brn3a-binding sequences in the TrkA minimal enhancer. The 5' protected sequence is 5'-TCTAAGAGATCTATTAATTTCTC-3' (C) and the 3' protected sequence is 5'-TCACCTAACTTATTCCAAGTGACATGCACACCCTCTTAA-3' (D). (E,F, left panels) Gel-shift assays showing that the protected sequences in (C,D) compete with the first and sixth 100 bp fragments in (A,B) for GST-Brn3a POU-domain fusion-protein binding. (E,F, top panels) Protected sequences from (C,D) share (red boxes) an A/T-rich core sequence. The mutant sequences used in the competitive gel-shift assays (left panels) and the following transgenic analysis (Figs 5, 6) are shown in red. (E,F, right panels) Competitive gel-shift assays using either the wild-type or mutated Brn3a-binding sites, showing that mutated Brn3a-binding sites do not bind the GST-Brn3a POU-domain fusion protein (lane 8) and, unlike wild-type sites (lanes 1-4), do not compete with wild-type sites for binding (lanes 5-7). Arrows in E,F indicate the DNA-protein complexes.