Regulatory DNAs may be missed

Nonconserved, noncoding regulatory sequences could be new mutation targets

By | March 24, 2006

Vast numbers of noncoding regulatory sequences may lurk in nonconserved DNA that are not detected by techniques typically used to identify these sequences, suggests research appearing online this week in Science. These findings have already triggered plans for subsequent investigations to uncover functional DNA that previous studies may have missed. "Functional noncoding sequences can exist below the radar of current predictions," coauthor Andrew McCallion told The Scientist. These sequences could prove novel targets for mutation and therapeutic screens, coauthor Shannon Fisher added. Current methods that identify functional noncoding DNA focus on comparing genomes to find conserved sequences, on the assumption that conservation over great evolutionary distances indicates selective pressure to preserve critical mechanisms. The researchers discovered that even though noncoding regions apparently are not conserved between the human developmental gene RET and its zebrafish counterpart ret, nearly all of the human noncoding regulatory sequences discovered at RET could also function when introduced into zebrafish. Using genomic sequence alignment tools such as AVID/VISTA, the researchers, all based at Johns Hopkins University in Baltimore, Md., compared the zebrafish ret locus with the orthologous interval in fugu pufferfish and generated 10 amplicons, representing 14 noncoding sequences conserved in zebrafish. They also compared the human RET locus with orthologous intervals in 12 nonhuman vertebrates and generated 13 amplicons, corresponding to 28 noncoding sequences conserved in humans. The researchers used a transposon-based transgenic assay to introduce the amplicons into zebrafish embryos. They found nine of the 10 zebrafish-derived amplicons triggered ret-specific reporter gene expression in the nervous, endocrine, and excretory systems. Additionally, 11 of the 13 human-derived amplicons could also drive expression in zebrafish embryos -- even in cells not present in mammals. Multiple sequences also appeared to trigger expression in the excretory system, despite the fact that fish and mammal systems differ both developmentally and anatomically. To test if their analyses missed conserved features between human and zebrafish sequences, the researchers repeated AVID/VISTA scans, reducing the window size down to 30 base pairs, and employed additional tests, as well. All failed to identify conserved sequences between the two species. This finding suggests functional components in conserved sequences are only four to 20 base pairs in size, too small for current in silico techniques to reliably detect. McCallion noted that sequence conservation often correctly points out functional DNA across both great and short evolutionary distances, as was the case with genome comparisons between zebrafish and fugu. "We simply contend that constraining the prediction of functional information based on conservation across great evolutionary distances probably causes you to miss a large amount of functional information." "This is a precise and well-executed exploration" into the presence of nonconserved noncoding regulatory sequences, Richard Gibbs at the Baylor College of Medicine, who did not participate in this study, told The Scientist. "It shows that we still have a lot to learn about the molecular evolution of gene regulatory elements," David Haussler at the University of California at Santa Cruz, also not a co-author, told The Scientist. Identifying such sequences will require high throughput functional assays such as one the team used, Edward Rubin at Lawrence Berkeley National Laboratory, who did not participate in this study, told The Scientist. Future investigations can reanalyze sequences surrounding any gene for regulatory potential, and the researchers have already received "dozens" of requests for collaborations, Fisher said. The more such sequences get discovered, "the more computational approaches can get refined to better identify them," she added. Researchers should also identify the transcription factors that bind to such elements, Fisher noted. Charles Choi Links within this article S. Fisher et al. "Conservation of RET Regulatory Function from Human to Zebrafish in the Absence of Sequence Conservation," Science, published online March 23, 2006. Andrew McCallion Shannon Fisher C. Holding. "Noncoding conservation," The Scientist, October 3, 2003. J.L. Peirce. "Following phylogenetic footprints," The Scientist, September 27, 2004. AVID/VISTA R. Robinson, ?Shadows provide illumination,? The Scientist, February 28, 2006. David Haussler C. Holding, ?Sequences come together,? The Scientist, March 31, 2004.

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