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（由申请人提供）： 二聚体 NF-κB 转录因子通过与位于靶基因启动子/增强子中的特定 DNA 序列（称为 κB 位点 DNA）结合来调节基因表达。这些二聚体可分为两组。含有转录激活结构域 (AD) 的 p50 和 p52 同二聚体不具有 AD，但与辅因子相关，它们可以参与转录。 Bcl3（p52：Bcl3 复合物）通过与两种类型的 kB 位点结合来参与转录。最近的实验表明，RelA 二聚体和 p52：Bcl3 复合物都可以通过与不同的 kB DNA 序列结合来激活和抑制转录。没有发现 NF-κB：κB DNA 复合物结合亲和力和转录输出之间的关系。该提案的重点是了解 κB 位点序列及其与 NF-κB 二聚体的结合机制之间的关系。我们渴望 NF-κB 二聚体与 κB 位点的结合动力学决定其是否充当转录抑制子或激活子。我们认为 NF-κB 与 DNA 的结合动力学与 NF-κB 的相互作用相耦合。我们将通过以下实验检验我们的假设：在目标 1 下，我们将研究 Bcl3 如何被脂多糖激活。 (LPS) 刺激以及它如何与 p52 同二聚体组装以与 kB 位点相互作用。我们将使用全基因组 ChIP-se 分析进行进一步测试，以强化该 NF-kB 复合物对两类不同类型的 kB 位点作用不同的说法。我们将进一步利用结构、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，我们将研究 kB 序列模式、RelA 之间的相关性。使用体外染色质模板的二聚体结合和转录输出将通过直接测量 NF-kB 二聚体与核小体结合的共激活子或辅阻遏物复合物的结合动力学来研究 NF-kB 二聚体如何找到嵌入单核小体模板中的 κB 位点。对于同一对 NF-kB 二聚体和 kB 位点，体外结合动力学与体内结合动力学相关。