Role of novel histone modifications and variants in transcriptional regulation

项目概要 核小体相关组蛋白的翻译后修饰 (PTM) 以及组蛋白变体 掺入，影响转录能力，并且在许多情况下已发现它们的失调 病理状态。组蛋白 H3 N 末端乙酰化、甲基化和磷酸化是常见的 PTM；的 这些 PTM 的精确组合可以调节染色质结构和基因组组织，从而导致 基因表达的变化。尽管为表征 H3 PTM 和 H3 变体付出了巨大的努力，但它们 机械和功能的相互作用，以及它们影响生物输出的能力，目前尚不清楚如何 组蛋白和许多组蛋白 PTM 整合来自上游信号级联的线索来调节基因表达。 我实验室的首要目标是定义调节组蛋白 PTM 模式和 揭示它们如何影响转录输出。在接下来的五年里，我们提出了一种组合方法 利用遗传、分子、细胞、生化和计算方法来定义新机制 其中一个人们知之甚少的组蛋白 H3 PTM，即 H3 苏氨酸 45 磷酸化 (pH3T45)，负责传递细胞信号 从上游激酶影响基因表达，并利用这种方法来描述新的致病性如何 组蛋白 H3 变异使表观基因组失调，从而改变细胞功能。我们的初步数据表明 H3T45 磷酸化状态 (1) 通过引导特定的 H3K4 修饰复合物来调节 H3K4 甲基化 染色质； (2) 通过竞争性组蛋白乙酰化/脱乙酰化破坏 H3K36me3； (3)调节RNA 通过与剪接因子和 RNA 外泌体复合物的差异关联进行加工。我们将剖析 H3K4-甲基转移酶复合物在结构、功能和生物输出方面有何不同 pH3T45 或未修饰的 H3T45。我们将描述 pH3T45 如何影响 H3K36 乙酰化的动力学和 DNA 修复背景下的 H3K36 甲基化。然后我们将定义 pH3T45 如何控制生产 通过检查未修饰的 H3T45 和 pH3T45 在整个 RNA 加工过程中的作用来分析成熟 RNA。最后，我们 已经鉴定出一系列与癌症相关的 H3 变体，其中氨基酸变为赖氨酸，称为 “H3 X 至 K”变体。我们的初步数据表明，H3 X 到 K 变异使近端 H3 PTM 失调 独特地调节基因表达。我们将利用我们研究 pH3T45 的经验来机械地 定义 H3 X 到 K 变体如何重新编程表观基因组以产生转录和功能性细胞 变化。这项研究将解决组蛋白 PTM 模式和 H3 变体的调控和影响 表达，这将让我们了解 H3 PTM 和变异表达如何成为人类疾病的基础。

PROJECT SUMMARY Posttranslational modification (PTM) of nucleosome-associated histone proteins, along with histone variant incorporation, influences transcriptional competence and their dysregulation has been identified in numerous pathological states. Histone H3 N-terminal acetylation, methylation, and phosphorylation are common PTMs; the precise combination of these PTMs can modulate chromatin architecture and genome organization, leading to changes in gene expression. Despite extensive efforts to characterize H3 PTMs and H3 variants, their mechanistic and functional interplay, and their ability to influence biological output, it remains unclear how histones and many histone PTMs integrate cues from upstream signaling cascades to regulate gene expression. The overarching objective for my laboratory is to define mechanisms that regulate histone PTM patterns and unmask how they influence transcriptional output. Over the next five years, we propose a combinatorial approach leveraging genetic, molecular, cellular, biochemical and computational methods to define novel mechanisms by which a poorly understood histone H3 PTM, H3 threonine 45 phosphorylation (pH3T45), relays cellular signals from upstream kinases to impact gene expression, and leverage this approach to delineate how novel pathogenic histone H3 variants dysregulate the epigenome to alter cellular function. Our preliminary data suggest that H3T45 phosphorylation status (1) modulates H3K4 methylation by directing specific H3K4-modifying complexes to chromatin; (2) disrupts H3K36me3 via competing histone acetylation/deacetylation; (3) regulates RNA processing through differential association with splicing factors and the RNA exosome complex. We will dissect how H3K4-methyltransferase complexes differ in structure, function, and biological output when associated with pH3T45 or unmodified H3T45. We will delineate how pH3T45 impacts the dynamics of H3K36 acetylation and H3K36 methylation in the context of DNA repair. We will then define how pH3T45 governs the production of mature RNA by examining the role of unmodified H3T45 and pH3T45 throughout RNA processing. Lastly, we have identified a series of cancer-associated H3 variants in which an amino acid is changed to a lysine, termed “H3 X to K” variants. Our preliminary data demonstrates that H3 X to K variants dysregulate proximal H3 PTMs to uniquely modulate gene expression. We will leverage our experience studying pH3T45 to mechanistically define how H3 X to K variants reprogram the epigenome to produce transcriptional and functional cellular changes. This research will address the regulation and effects of histone PTM patterns and H3 variant expression, which will inform our view of how H3 PTMs and variant expression underlies human disease.