Analysis of the RelA:CBP/p300 interaction reveals its involvement in NF-κB-driven transcription

• Published in: PLoS biology Vol. 11; no. 9; p. e1001647
• Main Authors: Mukherjee, Sulakshana P, Behar, Marcelo, Birnbaum, Harry A, Hoffmann, Alexander, Wright, Peter E, Ghosh, Gourisankar
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.09.2013; Public Library of Science (PLoS)
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; 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; 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; Promoter Regions, Genetic; Protein Binding; Protein Folding; Protein Structure, Tertiary; Transcription Factor RelA - genetics; Transcription Factor RelA - metabolism; Transcription, Genetic
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.1001647

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.