Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees
Adrien Chabot 1, Ralla Shrit 1, Ran Blekhman 1 and Yoav Gilad 1*
1 University of Chicago
* To whom correspondence should be addressed. E-mail: gilad{at}uchicago.edu.
Submitted on March 15, 2007
Revised on May 14, 2007
Accepted on 25 May 2007
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Abstract |
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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 are known. In order to identify differences in regulatory elements between human and chimpanzee, we focused on ten 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 ten 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 will provide a map of regulatory element differences between humans and our close evolutionary relatives.
Key Words:
Evolution, Gene-regulation, reporter-gene assays
