Investigation of gene regulation by NF-kappaB transcription factors

性状（由申请人提供）：二聚NF-？B转录因子通过与特定的DNA序列结合来调节基因的表达。B位点DNA，位于靶基因的启动子/增强子。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，我们没有发现任何相关性，链接NF-？B：？B DNA复合物结合亲和力和转录输出。这个建议的重点是了解之间的关系？B位点序列、其与NF-？B二聚体和转录输出。我们假设，NF-？B二聚体结合a？B位点决定它是作为转录的阻遏物还是激活物。我们建议，NF-？DNA的B与NF-？B与辅阻遏物和辅激活物的相互作用。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，动力学和突变的研究，以确定之间的相关性？B位点序列、结合动力学和转录潜力。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将研究如何NF-kB二聚体发现？通过直接测量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，确定嵌入单核小体模板中的B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。

DESCRIPTION (provided by applicant): Dimeric NF-?B transcription factors regulate gene expression by binding to specific DNA sequences, known as the ?B 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- ?B: ?B DNA complex binding affinity and transcriptional output. The focus of this proposal is to understand the relationship between ?B site sequence, its binding mechanism to NF-?B dimers, and transcriptional output. We hypothesize that the kinetics of NF-?B dimer binding to a ?B site determines whether it acts as a repressor or activator of transcription. We propose that the binding kinetics of NF-?B to DNA is coupled to NF-?B'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 ?B 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 ?B 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.