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Originally published as Genetics Published Articles Ahead of Print on June 11, 2007.

Genetics, Vol. 176, 2069-2076, August 2007, Copyright © 2007
doi:10.1534/genetics.107.073429

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Using Reporter Gene Assays to Identify cis Regulatory Differences Between Humans and Chimpanzees

Adrien Chabot, Ralla A. Shrit, Ran Blekhman and Yoav Gilad1

Department of Human Genetics, University of Chicago, Chicago, Illinois 60637

1 Corresponding author: Department of Human Genetics, University of Chicago, 920 E. 58th St., CLSC 325C, Chicago, IL 60637.
E-mail: gilad{at}uchicago.edu

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human–chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.


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