A substantial fraction of phenotypic differences between closely related species 
are likely caused by differences in gene regulation. While this has already been 
postulated over 30 years ago, only few examples of evolutionary changes in gene 
regulation have been verified. Here, we identified and investigated binding 
sites of the transcription factor GA-binding protein alpha (GABPa) aiming to 
discover cis-regulatory adaptations on the human lineage. By performing 
chromatin immunoprecipitation-sequencing experiments in a human cell line, we 
found 11,619 putative GABPa binding sites. Through sequence comparisons of the 
human GABPa binding regions with orthologous sequences from 34 mammals, we 
identified substitutions that have resulted in 224 putative human-specific GABPa 
binding sites. To experimentally assess the transcriptional impact of those 
substitutions, we selected four promoters for promoter-reporter gene assays 
using human and African green monkey cells. We compared the activities of 
wild-type promoters to mutated forms, where we have introduced one or more 
substitutions to mimic the ancestral state devoid of the GABPa consensus binding 
sequence. Similarly, we introduced the human-specific substitutions into 
chimpanzee and macaque promoter backgrounds. Our results demonstrate that the 
identified substitutions are functional, both in human and nonhuman promoters. 
In addition, we performed GABPa knock-down experiments and found 1,215 genes as 
strong candidates for primary targets. Further analyses of our data sets link 
GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and 
human-specific genes. Thus, we propose that differences in GABPa binding sites 
played important roles in the evolution of human-specific phenotypes.