Chromatin modifications that enhance DNA accessibility

项目摘要/摘要 提高DNA可及性的染色质修饰：真核DNA被紧密包装到 形成转录结构障碍的核小体，但RNA聚合酶有效地阅读 通过细胞内染色质的DNA。这一明显矛盾的关键是三磷酸腺苷不依赖的组蛋白 伴侣，包括异二聚体(促进染色质转录)复合体和 晶状体上皮源性生长因子单体蛋白与肝癌生长 因子2(HDGF2)。尽管FACT作为组蛋白伴侣的作用是公认的，但LEDGF和HDGF2 直到最近才被牵连为具有组蛋白伴侣活性。此外，LEDGF和HDGF2 也与调节人类免疫缺陷病毒1型(HIV-1)DNA整合有关 转化为染色质，其中LEDGF起主导作用，但其机制尚不清楚。我们 假设：(I)事实通过优先与核小体结合促进染色质重塑 被翻译后的组蛋白修饰破坏稳定，保留必要的组蛋白-DNA相互作用 用于核小体重组；(Ii)LEDGF和HDGF2作为阅读器蛋白，选择性地工作 通过它们优先结合到富含H3K36me2/3组蛋白修饰的区域，但具有 辅助域赋予的与事实的机械相似性；(Iii)通过它们的组蛋白伴侣 活性，LEDGF和HDGF2蛋白调节HIV-1DNA整合到活跃转录的基因中 以伴侣不稳定的H3K36me2/3富染色质为特征。我们提出两个目标： 目标1：定义事实对染色质状态的不同影响，以检验以下假设 FACT作为伴侣优先与未缠绕的染色质中间体和染色质结合 由于组蛋白修饰而不稳定，我们将使用光钳施加力来确定 平衡稳定性、波动开口率和核小体重新组装的能力 通过不稳定修饰的组蛋白在WT、突变体和截断事实存在时的破坏 酰化修饰。结果将揭示事实活动可以在多大程度上受到 不稳定的组蛋白修饰。目的2：确定LEDGF和HDGF2的作用机制 作为组蛋白伴侣和HIV-1整合的促进者。来检验LEDGF的假设 和HDGF2结合含H3K36me2/3的核小体，并介导核小体的拆解和 重组以及LEDGF和HDGF2在H3K36me2/3富集区引导HIV-1DNA整合 通过它们在这些部位的核小体伴侣活性，我们将测量野生型和 突变体LEDGF和HDGF2对核小体稳定性、动力学和重组的影响及其影响 关于HIV-1整合酶结合的研究。这一结果将决定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。

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.