Chromatin modifications that enhance DNA accessibility

项目总结/摘要 增强DNA可及性的染色质修饰：真核DNA被紧密包装成 核小体，形成转录的结构屏障，但RNA聚合酶有效地阅读 通过细胞中的染色质化DNA。这个明显矛盾的关键是ATP非依赖性组蛋白 分子伴侣，其包括异二聚体FACT（FACilitates染色质转录）复合物和 单体蛋白透镜上皮衍生生长因子（LEDGF）和肝癌衍生生长 因子2（HDGF 2）。尽管FACT作为组蛋白伴侣的作用已经得到了很好的证实，但LEDGF和HDGF 2 直到最近才被证实具有组蛋白伴侣活性。此外，LEDGF和HDGF 2 还涉及调节人类免疫缺陷病毒1型（HIV-1）DNA整合 在染色质中，LEDGF起主导作用，但其机制尚不清楚。我们 假设：（i）FACT通过优先结合核小体促进染色质重塑 通过翻译后组蛋白修饰使其不稳定，保留必要的组蛋白-DNA相互作用 （ii）LEDGF和HDGF 2作为阅读器蛋白，选择性地工作 通过它们优先结合到富含H3 K36 me 2/3组蛋白修饰的区域，但 辅助结构域赋予FACT的机制相似性;（iii）通过其组蛋白伴侣 活性，LEDGF和HDGF 2蛋白调节HIV-1 DNA整合到活跃转录的基因中 其特征是富含伴侣不稳定的H3 K36 me 2/3染色质。我们提出两个目标： 目的1：定义FACT对染色质状态的差异效应 FACT通过优先结合未缠绕的染色质中间体和染色质， 由于组蛋白修饰而不稳定，我们将用光学镊子施加力来确定 平衡稳定性，波动的开放率，以及核小体重组后的能力 在WT、突变体和截短的FACT存在下，通过去稳定化修饰的组蛋白的破坏 酰化修饰。结果将揭示FACT活性可以通过以下方式调节的程度： 破坏稳定的组蛋白修饰目的2：确定LEDGF和HDGF 2 作为组蛋白伴侣和HIV-1整合的促进者。为了验证LEDGF 和HDGF 2结合含H3 K36 me 2/3的核小体并介导核小体解体， LEDGF和HDGF 2在H3 K36 me 2/3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蛋白活性，我们将测量野生型和 突变LEDGF和HDGF 2对核小体稳定性、动力学和重组的影响及其作用 对HIV-1整合酶结合的影响本研究结果将进一步明确LEDGF和HDGF 2的作用机制 核小体伴侣活性和组蛋白甲基化在调节该活性中所起的作用。

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.