Chabot Adrien, Shrit Ralla A, Blekhman Ran, Gilad Yoav
Department of Human Genetics, University of Chicago, 920 E. 58th Street, Chicago, IL 60637, USA.
Genetics. 2007 Aug;176(4):2069-76. doi: 10.1534/genetics.107.073429. Epub 2007 Jun 11.
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.
人类与黑猩猩之间的大多数表型差异可能是由基因调控的差异导致的，而非蛋白质编码区域的变化。然而，迄今为止，仅鉴定出少数导致基因调控变化的人类 - 黑猩猩核苷酸差异。为了深入研究人类与黑猩猩之间调控元件的差异，我们聚焦于先前发现的在这两个物种间差异表达的10个基因。然后，我们针对这10个基因假定的人类和黑猩猩启动子设计了报告基因检测。在我们发现的7个在人类肝细胞系中具有活性的启动子中，人类和黑猩猩的启动子在4个案例中表现出显著不同的活性，其中3个案例重现了在微阵列实验中观察到的基因表达差异。因此，对于这3个基因，我们能够证明顺式作用元件的变化影响了人类与黑猩猩之间的表达差异。此外，通过对一个构建体（DDA3基因的启动子）进行定点诱变，我们能够鉴定出3个核苷酸，它们共同导致了物种间的顺式调控差异。这种方法的高通量应用可以提供人类与其近缘进化亲属之间调控元件差异的图谱。
