Identification and functional validation of candidate regulatory elements. (A) Representation of the upstream region of Kcnq1, indicating the fragments tested in the chromosome conformation capture (3C) assays. (B) Graphic display of the conservation profiles for the upstream region of Kcnq1. The base genome is mouse. Evolutionarily conserved regions (ECRs) of a minimum of 100 bp conserved above 70% sequence identity are displayed as red (intergenic) peaks, with the x-axis representing positions in the base genome and the y-axis representing percentage identity between the base and the aligned genomes. Annotated genes are depicted in blue. Numbered peaks represent the fragments tested for enhancer activity. (C) DNA sequences were inserted in pGL3-promoter vector upstream of the luciferase reporter. Luciferase activity was normalized to Renilla activity. All transfections were performed in triplicate. The asterisk denotes the interaction observed in neonatal heart by 3C. (D) Representative LacZ-stained embryos with in vivo enhancer activity. ECR4 and ECR2 exhibit tissue-specific activity, with numbers showing the reproducibility of LacZ reporter staining.