Comparative genomics of the SOX9 region in human and Fugu rubripes: Conservation of short regulatory sequence elements within large intergenic regions

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY s ex reversal, is caused by heterozygous mutations in and around the gene SOX 9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data fr om mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared similar to 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-cod ing genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene o rder in F. rubripes. In both species, very large intergenic distances separ ate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side o f SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. C omparative sequence analysis of the intergenic regions revealed five conser ved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available , mouse sequences confirm conservation of the elements. From the yeast arti ficial chromosome transgenic data, elements E3-E5 are candidate enhancers f or SOX9 expression in limb and vertebral column, and 8 of 10 CD translocati on breakpoints separate these elements from SOX9.