Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters

D1x homeobox genes of vertebrates are generally arranged as three bigene clusters on distinct chromosomes. The D1x1/D1x2, D1x5/D1x6, and D1x3/D1x7 clusters likely originate from duplications of an ancestral D1x gene pair. Overlaps in expression are often observed between genes from the different clusters. To determine if the overlaps are a result of the conservation of enhancer sequences between paralogous clusters, we compared the D1x1/2 and the D1x5/D1x6 intergenic regions from human, mouse, zebrafish, and from two pufferfish, Spheroides nephelus and Takifugu rubripes. Conservation between all five vertebrates is limited to four sequences, two in D1x1/D1x2 and two in D1x5/D1x6. These noncoding sequences are >75% identical over a few hundred base pairs, even in distant vertebrates. However, when compared to each other, the four intergenic sequences show a much more limited similarity. Each intergenic sequence acts as an enhancer when tested in transgenic animals. Three of them are active in the forebrain with overlapping patterns despite their limited sequence similarity. The lack of sequence similarity between paralogous intergenic regions and the high degree of sequence conservation of orthologous enhancers suggest a rapid divergence of D1x intergenic regions early in chordate/vertebrate evolution followed by fixation of cis-acting regulatory elements.