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：
10593119
负责人：
GOURISANKAR GHOSH
金额：
$ 31.38万
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593119
关键词：
Affect Affinity Binding Biochemical Biological Biology Biophysics Cartoons Cells Cellular Immunity Communication Complex Consensus DNA DNA Binding 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 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.

项目概要 NF-κB 二聚体与特定的 DNA 反应元件（称为 κB DNA 位点）结合，该元件位于数千个基因的启动子和增强子，并调节它们的表达。尽管大约 10 bp 长κB序列遵循共有序列，数百个特定序列可以符合该共有序列。变异可能导致 NF-κB-DNA 结合亲和力、动力学和构象的差异，从而导致事实上，我们报道过，只要一个碱基对的变化就可以改变转录输出。 NF-κB 二聚体对基因调控有严重影响 NF-κB:DNA 复合物的亲和力。体外实验并不总是与体内结合和基因调控相关。观察结果表明细胞中存在调节剂，但在体外并未包含它们另一方面，如果没有适当的体外实验，体内实验和体外实验结果不会一致。实验设置，对体内调节机制的正确研究是很难完成的。过去 10 年进行的细胞实验证实了其中几个的存在调节剂，但如果没有彻底的生化作用，就无法正确解释它们的作用机制我们将这些调节剂称为辅因子，这些辅因子改变 DNA 结合。 NF-κB p50:RelA 异二聚体和 RelA 同二聚体以 κB 序列特异性方式的亲和力。该提案的重点是使用新的实验提出一个统一原则，说明辅助因子如何调节 NF-κB 活性。我们提出，当 NF-κB:κB DNA 复合物之间的亲和力较弱时，辅因子可以发挥作用通过直接接触 NF-κB 而无需接触，积极增强 NF-κB:DNA 复合物的亲和力 DNA 允许基因表达发生。或者，辅因子可以通过去除 NF-κB 发挥负面作用 DNA（或降低亲和力）是已知的。研究其中一些辅助因子的体外作用模式具体来说，我们将确定其位点。 RelA 上正辅因子和负辅因子的相互作用以及它们如何利用变构机制来改变由于 RelA 的 DNA 结合尚无已知，因此我们还计划鉴定特定的辅因子。 p50 亚基并研究这些新辅因子是否以及如何与 RelA 特异性辅因子一起发挥作用。我们将生成辅因子结合缺陷的 RelA 突变体，并测试基因表达谱和细胞中的 DNA 结合会因特定刺激而改变。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated.

核因子γB 的RelA 同二聚体的DNA 结合亲和力和转录活性不相关。

DOI：
发表时间：
2017
期刊：
影响因子：
0
作者：
Mulero, Maria Carmen;Huang, De;Nguyen, H Thien;Wang, Vivien Ya;Li, Yidan;Biswas, Tapan;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar

Bcl3 Phosphorylation by Akt, Erk2, and IKK Is Required for Its Transcriptional Activity.

DOI：
10.1016/j.molcel.2017.06.011
发表时间：
2017-08-03
期刊：
Molecular cell
影响因子：
16
作者：
Wang VY;Li Y;Kim D;Zhong X;Du Q;Ghassemian M;Ghosh G
通讯作者：
Ghosh G

The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50.

先天免疫反应的特异性是通过 NF-κB p50 对干扰素反应元件的抑制来增强的。

DOI：
发表时间：
2011-02-22
期刊：
影响因子：
7.3
作者：
Cheng, Christine S;Feldman, Kristyn E;Lee, James;Verma, Shilpi;Huang, De;Huynh, Kim;Chang, Mikyoung;Ponomarenko, Julia V;Sun, Shao;Benedict, Chris A;Ghosh, Gourisankar;Hoffmann, Alexander
通讯作者：
Hoffmann, Alexander

A structural basis for selective dimerization by NF-κB RelB.

NF-κB RelB 选择性二聚化的结构基础。

DOI：
发表时间：
2013-06-12
期刊：
影响因子：
5.6
作者：
Vu, Don;Huang, De;Vemu, Annapurna;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar

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

RelA:CBP/p300 相互作用的分析揭示了它参与 NF-κB 驱动的转录。

DOI：
发表时间：
2013-09
期刊：
影响因子：
9.8
作者：
Mukherjee, Sulakshana P;Behar, Marcelo;Birnbaum, Harry A;Hoffmann, Alexander;Wright, Peter E;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar