Investigation of gene regulation by NF-kappaB transcription factors

描述（由申请人提供）：二聚体NF-κB转录因子通过结合特定的DNA序列来调节基因表达，这些DNA序列被称为κB位点DNA，位于目标基因的启动子/增强子中。这些二聚体可分为两类，一类含有转录激活域（transcription activation domain， AD），另一类不含。p50和p52同型二聚体不具有AD，但与辅助因子相关，它们可以参与转录。我们发现与Bcl3复合物的p52二聚体（p52:Bcl3复合物）通过结合两种类型的kB位点参与转录。最近的实验表明，RelA二聚体和p52:Bcl3复合物都可以通过结合不同的kB DNA序列来激活和抑制转录。到目前为止，我们还没有发现NF-κB: κB DNA复合物结合亲和力与转录输出之间的相关性。本研究的重点是了解κB位点序列及其与NF-κB二聚体的结合机制与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因子还是激活因子。我们认为NF-κB与DNA的结合动力学与NF-κB与辅抑制因子和辅激活因子的相互作用有关。我们将通过以下实验来验证我们的假设：在Aim 1中，我们将研究Bcl3如何被脂多糖（LPS）刺激激活，以及它如何与p52同型二聚体组装以与kB位点相互作用。我们将使用全基因组ChIP-se分析进一步测试，以加强两种不同类型的kB位点对NF-kB复合物的不同作用。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能之间的相关性。在Aim 2中，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将研究NF-kB二聚体如何通过直接测量NF-kB二聚体与核小体结合的辅激活物或辅抑制物复合物的结合动力学来找到嵌入在单核小体模板中的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。

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.