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

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

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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.

项目摘要 NF-κB二聚体与特定的DNA响应元件结合，称为位于该位置的κBDNA位点基因的启动子和增强子，并调节其表达。尽管大约10 bp 长κB序列遵循共识，数百个特定序列可以符合共识。顺序变化会导致NF-κB-DNA结合亲和力，动力学和构象的差异，导致转录输出的变化。确实，其他，我们报告说，只有一个BP更改可以 NF-κB二聚体对基因调节具有严重作用。 NF-κB的亲和力：衍生的DNA复合物在体外实验并不总是与体内结合和基因调节相关。这些观察结果表明，细胞中存在调节剂，并且不包含在体外体内和体外结果的实验不会和解。另一方面，没有适当的体外实验设置，在体内对监管机制的适当投资很难完成。在过去的10年中进行的细胞实验确定了其中几个此类的存在调节器，但是如果没有优先级的生化，无法正确解释其作用机制和生物物理实验。我们称这些调节器辅助因子。这些辅助因子改变了DNA结合 NF-κBp50的亲和力：以κB序列特异性方式的Rela异二聚体和RELA同型二聚体。这该提案的重点是使用新实验提出一个统一原则，以说明如何辅助因子调节NF-κB活性。我们建议，当NF-κB：κBDNA复合物之间的亲和力很弱时，辅助因子可以作用通过直接接触NF-κB而无需接触，从而积极增强NF-κB：DNA复合物的亲和力 DNA允许发生基因表达。另外，辅助因子可以通过删除NF-κB来负面作用 DNA（或减少亲和力）。已知几个阳性辅因子和很少的负辅因子。我们将在体外研究其中一些辅助因子的作用方式。具体来说，我们将确定阳性和负辅因子在Rela上的相互作用以及它们如何使用变构机制改变 Rela的DNA结合。由于没有p50特定的辅助因子，因此我们还计划识别特定的辅助因子到P50亚基，并研究这些新辅助因子是否以及如何与Rela特异性辅助因子一起起作用。我们将在辅因子结合中产生Rela有缺陷的突变体，并测试基因表达谱和如何细胞中的DNA结合对特定刺激的响应改变。