Journal Article

Analysis of the RelA:CBP/p300 Interaction Reveals Its Involvement in NF-κB-Driven Transcription

Behar, Marcelo, Mukherjee, Sulakshana P., Hoffmann, Alexander, Birnbaum, Harry A., Ghosh, Gourisankar, Wright, Peter E.

2013

Overview

NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components--first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: 3T3 Cells / Amino Acid Sequence / Animals / Binding proteins / Biology / Cell Differentiation - genetics / Cell Line / Cell Proliferation / E1A-Associated p300 Protein - genetics / E1A-Associated p300 Protein - metabolism / Gene Expression Profiling / Gene Expression Regulation - immunology / Genetic transcription / Hydrophobic and Hydrophilic Interactions / Magnetic Resonance Spectroscopy / Membrane Proteins - genetics / Membrane Proteins - metabolism / Mice / Molecular Sequence Data / NF-kappa B - genetics / NF-kappa B - metabolism / Phosphoproteins - genetics / Phosphoproteins - metabolism / Phosphorylation / Physiological aspects / Promoter Regions, Genetic / Protein Binding / Protein Folding / Protein Structure, Tertiary / Transcription Factor RelA - genetics / Transcription Factor RelA - metabolism / Transcription, Genetic