The landscape of NFκB transcription dynamics

NFκB 转录动力学景观

• 批准号: 10686820
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 56.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686820
• 关键词: Acceleration; Acquired Immunodeficiency Syndrome; Affinity; Apoptotic; Arthritis; Asthma; Atomic Force Microscopy; Autoimmune Diseases; Behavior; Binding; Biochemical; Biophysics; Cell Nucleus; Cells; Cessation of life; ChIP-seq; Code; Collaborations; Complex; Computer Models; Coupling; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Diabetes Mellitus; Disease; Elements; Endowment; Event; Excision; Exhibits; Family; Floods; Gene Expression; Genes; Genetic Enhancer Element; Genetic Transcription; Goals; Growth; I Kappa B-Alpha; Immune; Immune response; In Vitro; Inflammatory; Invaded; Kinetics; Laboratories; Length; Malignant Neoplasms; Measures; Mediating; Micro Array Data; Molecular; Monitor; Mutagenesis; Nuclear; Nuclear Export; Nuclear Proteins; Nucleosomes; Play; Process; Promoter Regions; Proteins; Publishing; RPS3 gene; Regulation; Relaxation; Research Personnel; Role; Series; Site; Slide; Speed; Stress; Structure; System; Systems Biology; TNF gene; TNFRSF5 gene; Testing; Therapeutic; Thermodynamics; Time; Transactivation; Transcription Coactivator; Transcription Initiation; Transcriptional Activation; Transcriptional Regulation; Viral; Virus Diseases; Work; biological adaptation to stress; biophysical techniques; cell growth; design; experimental study; flexibility; in vivo; molecular recognition; mutant; protein complex; protein folding; recruit; response; simulation; single-molecule FRET; stoichiometry; theories; transcription factor; transcriptome sequencing

Summary/Abstract The mechanism by which transcription factors assemble active transcription complexes on specific DNA sequences does not appear to follow a simple recognition code. Direct readout, wherein specific residues in the transcription factor “read” the specific DNA sequence through direct interactions is most often assumed to apply due to an oversimplified view of DNA as a rigid molecule. However, subtle, and not-so-subtle, structural changes occur when DNA binds to transcription factors. In addition, the DNA binding domains of transcription factors exhibit a large range of flexibility and often contain intrinsically disordered regions. These elements of flexibility endow the problem of transcription factor-DNA molecular recognition with many of the features of the protein folding problem. Our overall hypothesis is that transcription factor-DNA binding would instead be better described by similar principles as have been elucidated for the protein folding problem. Here, we will focus on the stress-response transcription factor, nuclear factor κB (NFκB), which activates hundreds of genes involved in growth regulation and the immune response. We will combine rigorous theory with molecular biophysical experiments to study the assembly kinetics of NFκB transcriptosome complexes. We will investigate coupling between DNA and NFκB as it relates to tandem κB sites, nucleosomal DNA, and the DNA-binding co-activator, RPS3. We predict that NFκB and additional nuclear proteins assemble into specific NFκB transcriptosomes on κB-DNA sites via a cooperative assembly process. We will test this hypothesis with the following aims: Aim 1 Determine the role of DNA context in NFκB binding. We will test the hypothesis that DNA context plays a key role in determining which NFκB binding events result in transcription activation by studying the binding of NFκB to a series of bona fide NFκB promoter and enhancer sequences both theoretically and experimentally. Aim 2 Explore how NFκB interacts with nucleosomal DNA and can invade or unwind nucleosomal DNA. We will test the hypothesis that NFκB is capable of disrupting nucleosome stability in a manner dependent on NFκB concentration and the sequence of DNA that is wrapped by the nucleosome, thereby exposing DNA for the initiation of transcription. Atomic force microscopy and computational modeling of the NFκB interaction with nucleosomes will be pursued. Aim 3 Determine how the ternary interaction between DNA, NFκB and the transcription co-activator, RPS3 forms. The NFκB coactivator, RPS3, associates with, and activates, subsets of NFκB transcription activation sites forming higher-order NFκB transcriptosome complexes. We will use the AWSEM-Suite code to predict the structures of these larger protein complexes and will verify the predicted long-range contacts between proteins and domains by NMR paramagnetic relaxation, SAXS, and HDX-MS experiments.

摘要/摘要 转录因子在特定DNA上组装活性转录复合物的机制 序列似乎不遵循简单的识别代码。直接读出，其中特定残基 转录因子通过直接相互作用“读取”特定的 DNA 序列通常被认为是 由于将 DNA 视为刚性分子的观点过于简单化而适用。然而，微妙的、不那么微妙的、结构性的 当 DNA 与转录因子结合时就会发生变化。此外，转录的 DNA 结合域 因素表现出很大的灵活性，并且通常包含本质上无序的区域。这些元素 灵活性赋予转录因子-DNA分子识别问题许多特征 蛋白质折叠问题。我们的总体假设是转录因子-DNA 结合会更好 通过与已阐明的蛋白质折叠问题类似的原理来描述。在这里，我们将重点关注 应激反应转录因子核因子 κB (NFκB)，可激活数百个相关基因 生长调节和免疫反应。我们将严谨的理论与分子生物物理学相结合 研究 NFκB 转录体复合物组装动力学的实验。我们将研究耦合 DNA 和 NFκB 之间的关系，因为它与串联 κB 位点、核小体 DNA 和 DNA 结合共激活剂有关， RPS3。我们预测 NFκB 和其他核蛋白组装成特定的 NFκB 转录体 通过协作组装过程的 κB-DNA 位点。我们将通过以下目标来检验这一假设： 目标 1 确定 DNA 背景在 NFκB 结合中的作用。我们将检验 DNA 背景发挥关键作用的假设 通过研究 NFκB 的结合来确定哪些 NFκB 结合事件导致转录激活 理论上和实验上一系列真正的 NFκB 启动子和增强子序列。目标2 探索 NFκB 如何与核小体 DNA 相互作用以及如何侵入或解开核小体 DNA。我们将测试 NFκB 能够以依赖于 NFκB 的方式破坏核小体稳定性的假设 浓度和被核小体包裹的 DNA 序列，从而暴露 DNA 转录的起始。 NFκB 相互作用的原子力显微镜和计算模型 将追寻核小体。目标 3 确定 DNA、NFκB 和 DNA 之间的三元相互作用如何 转录辅激活因子 RPS3 形式。 NFκB 共激活因子 RPS3 与 NFκB 的子集相关并激活 NFκB 转录激活位点形成高阶 NFκB 转录体复合物。我们将使用 AWSEM-Suite 代码可预测这些较大蛋白质复合物的结构，并验证预测的结果 通过 NMR 顺磁弛豫、SAXS 和 HDX-MS 确定蛋白质和结构域之间的长程接触 实验。