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; 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（由适用提供）：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。这些二聚体可以分为两组，一组包含转录激活域（AD），一个没有。 P50和P52同型二聚体不具有AD，而是与辅因子相关的，它们可以参与转录。我们发现，通过与两种类型的Kb位点结合，与Bcl3复合（p52：BCl3复合物）复合的p52同型二聚体参与了转录。最近的实验显示了Rela二聚体和p52：Bcl3复合物可以通过与不同的Kb DNA序列结合来激活和反映转录。到目前为止，我们发现尚未连接NF-κB：κBDNA复合结合亲和力和转录输出。该建议的重点是了解κB位点序列，其与NF-κB二聚体的结合机制和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它是转录的复制器还是激活因子。我们将通过以下实验检验我们的假设：在AIM 1下，我们将研究Bcl3如何通过脂糖糖（LPS）刺激激活Bcl3，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，以加强声称这两种不同类别的Kb站点Acre的作用不同，而NF-KB复合物的作用有所不同。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，结合动力学和转录电位之间的相关性。在AIM 2下，我们将使用体外染色质模板研究Kb序列模式，Rela Dimer结合和转录输出之间的相关性。 AIM 3将通过直接测量NF-kb二聚体与核小体结合的共激活因子或核心压轴络合物的结合动力学来研究NF-KB二聚体如何找到嵌入单核体模板中的κB位点。我们还将测试同一对NF-KB二聚体和Kb位点的体外结合动力学是否与体内结合动力学相关。