| Summary: | <p>Abstract</p> <p>Background</p> <p>Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent <it>in vitro </it>observations from the ENCODE project combined with <it>in vivo </it>analyses at the zebrafish <it>phox2b </it>locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations <it>in vivo </it>at the human <it>PHOX2B </it>locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data.</p> <p>Results</p> <p>Transposon-based transgenic analysis utilizing a tiling path proximal to human <it>PHOX2B </it>in zebrafish recapitulates the observations at the zebrafish <it>phox2b </it>locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish <it>phox2b </it>regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to <it>PHOX2B</it>, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our <it>in vivo </it>observations at <it>PHOX2B</it>. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert).</p> <p>Conclusion</p> <p>While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. <it>In vivo </it>observations at the <it>PHOX2B </it>locus, supported by our analyses of <it>in vitro </it>data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.</p>
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