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Cofactor-Mediated DNA Binding by the NF-kappaB Dimers

NF-kappaB 二聚体辅助因子介导的 DNA 结合

基本信息

批准号：
9887959
负责人：
GOURISANKAR GHOSH
金额：
$ 31.44万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9887959
关键词：
Affect Affinity Binding Biochemical Biological Biology Biophysics Cartoons Cells Cellular Immunity Communication Complex Consensus DNA DNA Binding DNA Sequence Data Disease Dissociation Enhancers Equilibrium Family Gene Activation Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Genomic DNA Glean Goals IRF3 gene Immunity In Vitro Inflammation Inflammatory Interleukin-1 Interleukins Investigation Kinetics Knowledge Mediating Modeling Molecular Molecular Conformation NF-kappa B Nuclear Proteins Output Process Proteins Regulator Genes Reporting Research Research Personnel Response Elements Retinoblastoma Retinoblastoma Protein Ribosomal Proteins SRC-associated p68 protein Site Solid Specificity Stimulus TNFRSF5 gene TP53 gene Testing Transcriptional Regulation Tumor Suppressor Proteins Variant Work cofactor combat dimer experimental study genome-wide in vivo insight link protein member mutant negative affect novel therapeutic intervention peptidomimetics programs promoter recruit response retinoblastoma tumor suppressor transcription factor

项目摘要

Project Summary The NF- κB dimers bind to specific DNA response elements known as the κB DNA sites located in the promoters and enhancers of thousands of genes, and regulate their expression. Although the roughly 10 bp long κB sequences follow a consensus, hundreds of specific sequences can fit the consensus. Sequence variations can result in differences in NF-κB-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 have severe effect in gene regulation by the NF-κB dimers. Affinities of the NF-κB:DNA complexes derived 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-κB p50:RelA heterodimer and RelA homodimers in a κB sequence-specific manner. The focus of this proposal is to use new experiments to propose a unifying principle of how the cofactors regulate NF-κB activity. We propose that when the affinity between an NF-κB:κB DNA complex is weak, a cofactor can act positively enhancing the affinity of NF-κB:DNA complexes by directly contacting NF-κB without contacting DNA allowing gene expression to occur. Alternatively, a cofactor can act negatively by removing NF-κB 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 interaction of both positive and negative cofactors on RelA and how they use allosteric mechanism to alter DNA binding by RelA. Since no cofactor specific to p50 is known, we also plan to identify cofactors specific to the p50 subunit and investigate if and how these new cofactors act together with RelA-specific cofactors. We will generate mutants of RelA defective in cofactor binding and test how gene expression profile and DNA binding in cells alters in response to specific stimulus.

项目总结