Specificity of Transcription Activation by NF-kB Subunits

Citation

Leung, Thomas Hin-Chai (2005) Specificity of Transcription Activation by NF-kB Subunits. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/100y-3c61. https://resolver.caltech.edu/CaltechETD:etd-10042004-090452

Abstract

The transcription factor NF-kappaB is a regulator of a wide variety of processes including inflammation, innate and adaptive immunity, apoptosis, and learning. How can one factor be accurately involved in so many different processes and generate without exception precise and appropriate responses? Four members of the NF-kappaB transcription factor family are involved in gene activation, and they hetero- or homodimerize with each other to bind DNA. This allows for many different potential combinations of NF-kappaB dimers. To test whether NF-kappaB-dependent genes require specific NF-kappaB family members for gene activation, cell lines were generated lacking in individual and multiple NF-kappaB proteins. Using TNFalpha as an inducer, a panel of endogenous NF-kappaB responsive genes showed a wide range of subunit specificities. Given that the NF-kappaB consensus binding site sequence is very broad and that crystal structures of NF-kappaB have not identified enough dimer-specific DNA-binding contacts to rationalize specific NF-kappaB binding sites, kappaB sites were compared from a single gene to another and no direct correlation was found between kappab site sequence and kappaB family member requirements. However when interspecies comparisons were made of the same gene, a remarkable constancy of the kappaB site sequence was found, which suggested that individual sites have important functional characteristics. To test this theory, a novel lentiviral system was created that incorporated regulatory sequences into cellular DNA. Then by simply swapping sites between kappaB-dependent genes, NF-kappaB dimer specificity of the promoters was altered and revealed that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which co-activators will form productive interactions with the bound kappaB dimer. My findings suggest that a particular DNA-binding site may impart a specific configuration to bound transcription factors that specifies the requirement for particular co-activators. Taken together, I have taken the first steps to dissecting how the promoter code influences individual NF-kappaB family members to function on NF-kappaB responsive genes and to regulate gene expression.

Thesis Files