Investigation of gene regulation by NF-kappaB transcription factors

NF-κB转录因子基因调控的研究

• 批准号: 8904029
• 负责人: GOURISANKAR GHOSH
• 金额: $ 31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904029
• 关键词: 3T3 Cells; Address; Affinity; Binding; Biochemical; Biological Process; Bone Marrow; Cells; ChIP-seq; Chromatin; Classification; Code; Communication; Complex; Consensus; Coupled; DNA; DNA Binding; DNA Sequence; DNA-Directed RNA Polymerase; Data; EP300 gene; Enhancers; Eukaryota; Fibroblasts; Formaldehyde; Funding; Gene Expression; Gene Expression Regulation; Gene Targeting; General Transcription Factors; Genes; Genetic Transcription; Health; Histones; In Vitro; Investigation; Kinetics; Laboratories; Lead; Length; Link; Measures; Mediating; Mediator of activation protein; Methods; Monitor; NF-kappa B; Nucleosomes; Output; Pattern; Polysaccharides; Positioning Attribute; Process; Proteins; Recruitment Activity; Regulation; Reporter; Repression; Research; Response Elements; Signal Transduction; Site; Specificity; System; TNFRSF5 gene; Testing; Time; Transcription Coactivator; Transcriptional Activation; Transcriptional Activation Domain; Transferase; Tumor Necrosis Factor-alpha; base; cofactor; crosslink; dimer; gene repression; genome-wide; in vitro testing; in vivo; macrophage; programs; promoter; research study; structural biology; transcription factor

DESCRIPTION (provided by applicant): Dimeric NF-κB transcription factors regulate gene expression by binding to specific DNA sequences, known as the κB site DNA, located in the promoter/enhancer of target genes. These dimers can be classified into two groups, one containing the transcription activation domain (AD) and one without. p50 and p52 homodimers do not possess AD but in association with cofactors they can participate in transcription. We found that the p52 homodimer complexed to Bcl3 (p52:Bcl3 complex) is involved in transcription by binding to two types of kB sites. Recent experiments have shown the both RelA dimers and p52:Bcl3 complex can both activate and repress transcription by binding to distinct kB DNA sequences. So far we found no correlation that links NF-κB: κB DNA complex binding affinity and transcriptional output. The focus of this proposal is to understand the relationship between κB site sequence, its binding mechanism to NF-κB dimers, and transcriptional output. We hypothesize that the kinetics of NF-κB dimer binding to a κB site determines whether it acts as a repressor or activator of transcription. We propose that the binding kinetics of NF-κB to DNA is coupled to NF-κB's interactions with corepressors and coactivators. We will test our hypothesis through the following experiments: Under Aim 1, we will investigate how Bcl3 is activated by lipo-polysaccharide (LPS) stimulation and how it assembles with p52 homodimer to interact with kB sites. We will further test using genome-wide ChIP-se analysis to strengthen out claim that two distinct classes of kB sites acre acted differently by this NF-kB complex. We will further use structural, kinetic, and mutational studies to determine the correlation between κB site sequence, binding kinetics, and transcriptional potential. Under Aim 2, we will investigate the correlation between kB sequence pattern, RelA dimer binding and transcriptional output using in vitro chromatin templates. Aim 3 will investigate how NF-kB dimers find κB sites embedded in mononucleosome templates by directly measuring binding kinetics of NF-kB dimer to nucleosome bound coactivator or corepressor complexes. We will also test if in vitro binding kinetics correlates with in vivo binding kinetics for the same pair of NF-kB dimer and kB site.

描述（由申请方提供）：二聚体NF-κB转录因子通过与特定DNA序列（称为κB位点DNA，位于靶基因的启动子/增强子中）结合来调节基因表达。这些二聚体可以分为两组，一组含有转录激活结构域（AD），另一组不含。p50和p52同源二聚体不具有AD，但与辅因子相关，它们可以参与转录。我们发现，p52同源二聚体复合Bcl 3（p52：Bcl 3复合物）参与转录结合两种类型的kB位点。最近的实验表明，RelA二聚体和p52：Bcl 3复合物都可以通过结合不同的kB DNA序列来激活和抑制转录。到目前为止，我们没有发现NF-κB：κB DNA复合物结合亲和力与转录输出之间的相关性。该提案的重点是了解κB位点序列、其与NF-κB二聚体的结合机制以及转录输出之间的关系。我们假设NF-κB二聚体与κB位点结合的动力学决定了它是作为转录的抑制剂还是激活剂。我们认为NF-κB与DNA的结合动力学与NF-κB与辅阻遏物和辅激活物的相互作用有关。我们将通过以下实验来验证我们的假设：在目标1下，我们将研究Bcl 3如何被脂多糖（LPS）刺激激活以及它如何与p52同源二聚体组装以与kB位点相互作用。我们将进一步测试使用全基因组ChIP-se分析，以加强我们的主张，即两种不同类别的kB位点英亩不同地发挥作用，这种NF-κ B复合物。我们将进一步使用结构、动力学和突变研究来确定κB位点序列、结合动力学和转录潜力之间的相关性。在目标2下，我们将使用体外染色质模板研究kB序列模式、RelA二聚体结合和转录输出之间的相关性。目的3通过直接测定NF-κ B二聚体与核小体结合的辅激活因子或辅阻遏因子复合物的结合动力学，研究NF-κ B二聚体如何在核小体模板中找到κB位点。我们还将测试体外结合动力学是否与同一对NF-κ B二聚体和κ B位点的体内结合动力学相关。