Fig. 4. Binding of NKX2.1 to a conserved consensus binding sequence in the Lhx6 promoter. (A) Evolutionarily conserved region (ECR) visualization of the LHX6 gene locus in the human genome. The conservation profile of the human sequence in comparison with the mouse, chicken, frog and fugu genomes is shown. In the model, the Lhx6 loci of the given species are displayed on the horizontal axis and the vertical axis represents the percentage of base-pair identity (from 50 to 100%) between the given species and human. The horizontal bar above each species provides an overview of the distribution of ECRs. A conserved alignment is blue if it overlaps with a coding exon. Yellow, untranslated region; pink, intron (although most of the pink region 5' to the Lhx6 translation start site in fact appears to be an intergenic region, see NCBI sequence data); red, intergenic region. The green bars at the bottom indicate repetitive elements in the sequence. (B) rVISTA analysis revealed a conserved NKX2.1/TITF1 binding site 240 bp from the translation start site. The arrowheads indicate the locations of the PCR primers used to clone a 2.1 kb promoter fragment of genomic DNA (see Materials and methods and Figs 5, 6). (C) The NKX2.1 consensus binding sequence is located within a 119 bp PCR product that was used to probe chromatin immunoprecipitation results on E12.5 MGE. IgG, control mouse IgG; Nkx2.1 Ab, mouse anti-NKX2.1; Input, control PCR on a crosslinked, sonicated MGE sample; left, molecular weight marker. These results suggest that NKX2.1 binds the Lhx6 promoter at a highly conserved NKX2.1 consensus binding sequence in vivo.