title: Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.
abstract: 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.
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Vasundhara
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Jordan
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Yari
authors.initials: Y
authors.lastname: Ciribilli
authors.affiliation: Chromosome Stability Group; National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bisio
authors.affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics; D061986:MCF-7 Cells; D016328:NF-kappa B / Q000235:genetics*; D052002:NF-kappa B p50 Subunit / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D051996:Transcription Factor RelA / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D015003:Yeasts / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D004247:DNA