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The identification of enhancers with predicted specificities in vertebrate genomes remains a significant challenge that is hampered by a lack of experimentally validated training sets. In this study, we leveraged extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements and characterized the in vivo enhancer activity of human-fish conserved and ultraconserved1 noncoding elements on human chromosome 16 as well as such elements from elsewhere in the genome. We initially tested 165 of these extremely conserved sequences in a transgenic mouse enhancer assay and observed that 48 percent (79/165) functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While driving expression in a broad range of anatomical structures in the embryo, the majority of the 79 enhancers drove expression in various regions of the developing nervous system. Studying a set of DNA elements that specifically drove forebrain expression, we identified DNA signatures specifically enriched in these elements and used these parameters to rank all ~;3,400 human-fugu conserved noncoding elements in the human genome. The testing of the top predictions in transgenic mice resulted in a three-fold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of in vivo-characterized human gene enhancers and illustrate the future utility of such training sets for a variety of iological applications including decoding the regulatory vocabulary of the human genome.

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intra-species sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents and a set of genomic intervals amplified, resequenced and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom and raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species. The sequence data from this study has been submitted to GenBank under accession nos. AY667278-AY667407.