The NF- ?B dimers bind to specific DNA response elements known as the ?B DNA sites located in the promotersandenhancersofthousandsofgenes,andregulatetheirexpression.Althoughtheroughly10bp long ?B sequences follow a consensus,hundreds of specific sequences can fit the consensus. Sequence variations can result in differences in NF-?B-DNA binding affinity, kinetics and conformations leading to changes in transcriptional output. Indeed, other and we reported that as few as a single bp change can havesevereeffectingeneregulationbytheNF-?Bdimers.AffinitiesoftheNF-?B:DNAcomplexesderived from in vitro experiments do not always correlate with in vivo binding and gene regulation. These observations suggest that there are modulators present in the cell and without their inclusion in in vitro experiments in vivo and in vitro results will not reconcile. On the other hand, without proper in vitro experimental set up, proper investigation of regulatory mechanisms in vivo is difficult to accomplish. Cellular experiments performed over the past 10 years established the presence of several of such modulators, but their mechanisms of action could not be properly explained without thorough biochemical and biophysical experiments. We term these modulators cofactors. These cofactors alter the DNA binding affinity of NF-?B p50:RelA heterodimer and RelA homodimers in a ?B sequence-specific manner. The focus of this proposal is to use new experiments to propose a unifying principle of how the cofactors regulateNF-?Bactivity. We propose that when the affinity between an NF-?B:?B DNA complex is weak, a cofactor can act positively enhancing the affinity of NF-?B:DNA complexes by directly contacting NF-?B without contacting DNA allowing geneexpression tooccur.Alternatively,a cofactor canact negatively by removing NF-?B off the DNA (or reduce affinity). Several positive cofactors and few negative cofactors are known. We will investigate the mode of actions of a few of these cofactors in vitro. Specifically, we will identify the site of interactionofboth positive and negative cofactors on RelAandhowtheyuse allosteric mechanismtoalter DNAbindingbyRelA.Sincenocofactorspecifictop50isknown,wealsoplantoidentifycofactorsspecific tothep50subunitandinvestigateifandhowthesenewcofactorsacttogetherwithRelA-specificcofactors. We will generate mutants of RelA defective in cofactor binding and test how gene expression profile and DNAbindingincellsaltersinresponsetospecificstimulus.

Public Health Relevance

Transcriptionofeukaryoticgenesisahighlycomplexprocessthatrequirescommunicationbetweenthe promoter DNA and sequence-specific DNA binding transcription factors such as NF-kappaB (NF-?B) dimers. Our proposed research plans to uncover how cofactors regulate the DNA binding of NF-?B RelAdimers,andhowintheabsenceofcofactorstherecruitmentofRelAingenepromotersisaffected leadingtodefectivebiologicalprograms.ThisproposalplanstoidentifyNF-?Bsubunit-specificcofactors andinvestigatethedetailedmechanismofcofactor-assistedDNAbindingofNF-?Bdimers.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Adkins, Ronald
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University of California, San Diego
Schools of Arts and Sciences
La Jolla
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Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo et al. (2018) Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor ?B RelA Subunit. Biochemistry 57:2943-2957
Mulero, Maria Carmen; Huang, De-Bin; Nguyen, H Thien et al. (2017) DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor ?B are not correlated. J Biol Chem 292:18821-18830
Wang, Vivien Ya-Fan; Li, Yidan; Kim, Daniel et al. (2017) Bcl3 Phosphorylation by Akt, Erk2, and IKK Is Required for Its Transcriptional Activity. Mol Cell 67:484-497.e5
Polley, Smarajit; Passos, Dario Oliveira; Huang, De-Bin et al. (2016) Structural Basis for the Activation of IKK1/?. Cell Rep 17:1907-1914
Mukherjee, Sulakshana P; Behar, Marcelo; Birnbaum, Harry A et al. (2013) Analysis of the RelA:CBP/p300 interaction reveals its involvement in NF-?B-driven transcription. PLoS Biol 11:e1001647
Vu, Don; Huang, De-Bin; Vemu, Annapurna et al. (2013) A structural basis for selective dimerization by NF-?B RelB. J Mol Biol 425:1934-1945
Ghosh, Gourisankar; Wang, Vivien Ya-Fan; Huang, De-Bin et al. (2012) NF-?B regulation: lessons from structures. Immunol Rev 246:36-58
Wang, Vivien Ya-Fan; Huang, Wendy; Asagiri, Masataka et al. (2012) The transcriptional specificity of NF-?B dimers is coded within the ?B DNA response elements. Cell Rep 2:824-39
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Cheng, Christine S; Feldman, Kristyn E; Lee, James et al. (2011) The specificity of innate immune responses is enforced by repression of interferon response elements by NF-?B p50. Sci Signal 4:ra11

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