Efficient identification of regulatory sequences in the chicken genome by a powerful combination of embryo electroporation and genome comparison

Recently expanded knowledge of gene regulation clearly indicates that the regulatory sequences of a gene, usually identified as enhancers, are widely distributed in the gene locus, revising the classical view that they are clustered in the vicinity of genes. To identify regulatory sequences for Sox2 expression governing early neurogenesis, we scanned the 50-kb region of the chicken Sox2 locus for enhancer activity utilizing embryo electroporation, resulting in identification of a number of enhancers scattered throughout the analyzed genomic span. The 'pan-neural' Sox2 expression in early embryos is actually brought about by the composite activities of five separate enhancers with distinct spatio-temporal specificities. These and other functionally defined enhancers exactly correspond to extragenic sequence blocks that are conspicuously conserved between the chicken and mammalian genomes and that are embedded in sequences with a wide range of sequence conservation between humans and mice. The sequences conserved between amniotes and teleosts correspond to subregions of the enhancer subsets which presumably represent core motifs of the enhancers, and the limited conservation partly reflects divergent expression patterns of the gene. The phylogenic distance between the chicken and mammals appears optimal for identifying a battery of genetic regulatory elements as conserved sequence blocks, and chicken embryo electroporation facilitates functional characterization of these elements.