Chromatin modifications that enhance DNA accessibility

染色质修饰可增强 DNA 可及性

基本信息

批准号：
10718867
负责人：
MARK C WILLIAMS
金额：
$ 52.9万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10718867
关键词：
Acylation Affect Antiviral Agents Atomic Force Microscopy Behavior Binding Biological Process Cells Charge Chromatin Complex Crystallins DNA DNA Integration DNA Packaging DNA-Directed RNA Polymerase Data Development Disease Dissociation Equilibrium Excision Fluorescence Genes Genetic Transcription Growth Factor HIV-1 Histones Individual Instruction Integrase Kinetics Learning Lysine Measures Mediating Metabolic Methods Modification Molecular Molecular Chaperones Nucleosomes Organism Physiological Play Process Proteins RNA Reader Role Site Specificity Stress Testing Time Virus Replication cancer cell cancer therapy chromatin modification chromatin remodeling hepatoma-derived growth factor histone methylation histone modification laser tweezer lens epithelium-derived growth factor mimetics monomer mutant optic tweezer preservation single molecule tool

项目摘要

Project Summary/Abstract Chromatin modifications that enhance DNA accessibility: Eukaryotic DNA is tightly packaged into nucleosomes, which form structural barriers to transcription, yet RNA polymerases effectively read through chromatinized DNA in cells. Crucial to this apparent paradox are ATP-independent histone chaperones, which include the heterodimeric FACT (FAcilitates Chromatin Transcription) complex and the monomeric proteins lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2). Whereas FACT’s role as a histone chaperone is well-established, LEDGF and HDGF2 were only recently implicated as having histone chaperone activity. Furthermore, LEDGF and HDGF2 have also been implicated in modulating human immunodeficiency virus type 1 (HIV-1) DNA integration into chromatin, with LEDGF playing a dominant role, but the mechanisms remain unclear. We hypothesize that: (i) FACT facilitates chromatin remodeling by preferentially binding to nucleosomes destabilized by post-translational histone modifications, preserving histone-DNA interactions necessary for nucleosome reassembly; (ii) LEDGF and HDGF2 function as reader proteins, working selectively through their preferential binding to regions rich in H3K36me2/3 histone modifications, but have mechanistic similarities to FACT imparted by auxiliary domains; (iii) through their histone chaperone activity, LEDGF and HDGF2 proteins modulate HIV-1 DNA integration into actively transcribed genes characterized by chaperone-destabilized H3K36me2/3-rich chromatin. We propose two aims: Aim 1: Define the differential effects of FACT on the chromatin state To test the hypothesis that FACT acts as a chaperone by preferentially binding to unwound chromatin intermediates and chromatin destabilized by histone modifications, we will apply forces with optical tweezers to determine the equilibrium stability, fluctuational opening rate, and the ability of nucleosomes to reassemble after disruption in the presence of WT, mutant, and truncated FACT for histones modified through destabilizing acylation modifications. The results will reveal the extent to which FACT activity can be regulated by destabilizing histone modifications. Aim 2: Determine the mechanisms by which LEDGF and HDGF2 act as histone chaperones and facilitators of HIV-1 integration. To test the hypotheses that LEDGF and HDGF2 bind H3K36me2/3-containing nucleosomes and mediate nucleosome disassembly and reassembly and that LEDGF and HDGF2 direct HIV-1 DNA integration at H3K36me2/3-enriched loci through their nucleosome chaperone activity at these sites, we will measure the effects of wild type and mutant LEDGF and HDGF2 on nucleosome stability, dynamics and reassembly, as well as their effects on HIV-1 integrase binding. The results will determine the mechanism of LEDGF and HDGF2 nucleosome chaperone activity and the role played by histone methylation in regulating that activity.

项目概要/摘要增强 DNA 可及性的染色质修饰：真核 DNA 被紧密包装在核小体，形成转录的结构障碍，但 RNA 聚合酶可以有效地读取这种明显悖论的关键是不依赖于 ATP 的组蛋白。分子伴侣，包括异二聚体 FACT（促进染色质转录）复合物和单体蛋白晶状体上皮源性生长因子 (LEDGF) 和肝癌源性生长 FACT、LEDGF 和 HDGF2 之间的因子 2 (HDGF2) 作为组蛋白伴侣的作用是明确的。最近才发现 LEDGF 和 HDGF2 具有组蛋白伴侣活性。还涉及调节人类免疫缺陷病毒 1 型 (HIV-1) DNA 整合 LEDGF 发挥主导作用，但其机制仍不清楚。提出：（i）FACT 通过优先结合核小体促进染色质重塑因翻译后组蛋白修饰而不稳定，保留必要的组蛋白-DNA 相互作用用于核小体重组；(ii) LEDGF 和 HDGF2 作为读取蛋白，选择性地发挥作用通过它们优先结合富含 H3K36me2/3 组蛋白修饰的区域，但辅助结构域与 FACT 的机制相似性 (iii) 通过其组蛋白伴侣 LEDGF 和 HDGF2 蛋白调节 HIV-1 DNA 整合到活跃转录的基因中以分子伴侣不稳定的 H3K36me2/3 丰富染色质为特征，我们提出了两个目标：目标 1：定义 FACT 对染色质状态的不同影响检验以下假设： FACT 通过优先结合未缠绕的染色质中间体和染色质来充当伴侣由于组蛋白修饰而不稳定，我们将用光镊施加力来确定平衡稳定性、波动开放率以及核小体重组后的能力在 WT、突变体和截短 FACT 存在的情况下，通过不稳定修饰修饰的组蛋白会受到破坏结果将揭示 FACT 活性受调节的程度。目标 2：确定 LEDGF 和 HDGF2 的机制。作为 HIV-1 整合的组蛋白伴侣和促进剂检验 LEDGF 的假设。 HDGF2 结合含有 H3K36me2/3 的核小体并介导核小体分解和重组以及 LEDGF 和 HDGF2 在 H3K36me2/3 富集位点指导 HIV-1 DNA 整合通过它们在这些位点的核小体伴侣活性，我们将测量野生型和突变体 LEDGF 和 HDGF2 对核小体稳定性、动力学和重组及其影响 HIV-1整合酶结合的结果将确定LEDGF和HDGF2的机制。核小体伴侣活性以及组蛋白甲基化在调节该活性中所起的作用。