Figure 7
Brn3a binds to regulatory sequences within the Runx3 locus. (A) Map of a 300-kb region of mouse chromosome 4 including the Runx3 and Syf2 loci. The location of three sites (-173 kb, -94 kb and -25 kb) showing strong in vitro Brn3a binding are indicated (blue circles). Also shown is an interspecies sequence alignment of a region within the proposed -94-kb enhancer, which contains a conserved consensus Brn3a binding site (blue box) and additional conserved TAAT motifs (green boxes) that represent additional potential homeodomain transcription factor binding sites. (B) EMSA for Brn3a binding to sites identified by sequence search; assays for the three sites exhibiting strongest binding are shown. P, probe only; -, probe incubated with protein extract from untransfected HEK cells; +, probe incubated with protein extract from HEK cells transfected with a Brn3a expression construct; Ab, Brn3a-transfected cell extract plus supershifting anti-Brn3a antibody; arrow, shifted band; asterisk, supershifted band. Multiple shifted bands may indicate cooperative binding of Brn3a to adjacent sites [56]. (C) ChIP profiling of in vivo Brn3a binding to the Runx3 locus in E13.5 TG. Two or three primer pairs were made to the regions containing each of the nine potential Brn3a binding sites identified by sequence search (Additional file 4). Quantitative PCR assays were performed on immunoprecipitated chromatin selected with an anti-Brn3a antibody from three independent samples. Fold enrichment for selected/unselected was calculated using the cycle threshold difference method (Methods). Graph shows the mean + standard error for three assays of independent samples. As expected, binding is not observed at the promoter region of the Alb (serum albumin) locus, which is not transcribed in neurons and does not contain Brn3a binding sites [14]. Unpaired t-test for selection at -94-kb site: n = 8 experimental assays, n = 13 alb locus controls, P = 7 × 10-5. (D) ChIP profiling of histone H3 acetylation at the Runx3 locus. Assays were performed as described in (C), with an antibody recognizing lysine 9/14 acetyl histone H3. Increased acetylation in the vicinity of the Brn3a binding site at -94 kb suggests an open conformation of chromatin at this location.