The NF-κB transcription factor family plays a central role in innate immunity 
and inflammation processes and is frequently dysregulated in cancer. We 
developed an NF-κB functional assay in yeast to investigate the following 
issues: transactivation specificity of NF-κB proteins acting as homodimers or 
heterodimers; correlation between transactivation capacity and in vitro DNA 
binding measurements; impact of co-expressed interacting proteins or of small 
molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 
cDNAs were cloned into centromeric expression vectors under inducible GAL1 
promoter in order to vary their expression levels. Since p50 lacks a 
transactivation domain (TAD), a chimeric construct containing the TAD derived 
from p65 was also generated (p50TAD) to address its binding and transactivation 
potential. The p50TAD and p65 had distinct transactivation specificities towards 
seventeen different κB response elements (κB-REs) where single nucleotide 
changes could greatly impact transactivation. For four κB-REs, results in yeast 
were predictive of transactivation potential measured in the human MCF7 cell 
lines treated with the NF-κB activator TNFα. Transactivation results in yeast 
correlated only partially with in vitro measured DNA binding affinities, 
suggesting that features other than strength of interaction with naked DNA 
affect transactivation, although factors such as chromatin context are kept 
constant in our isogenic yeast assay. The small molecules BAY11-7082 and 
ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in 
yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate 
NF-κB features found in human cells, thereby providing opportunities to address 
various NF-κB functions, interactions and chemical modulators.