Role of novel histone modifications and variants in transcriptional regulation

项目总结 核小体相关组蛋白及其变异体的翻译后修饰 整合，影响转录能力和它们的失调已被发现在许多 病态。组蛋白H3N-末端乙酰化、甲基化和磷酸化是常见的PTM； 这些PTM的精确结合可以调节染色质结构和基因组组织，导致 基因表达的变化。尽管对H3PTM和H3变种的特征进行了广泛的努力，但他们的 机制和功能的相互作用，以及它们影响生物产量的能力，目前尚不清楚 组蛋白和许多组蛋白PTM整合来自上游信号级联的信号来调节基因表达。 我的实验室的首要目标是定义调节组蛋白PTM模式和 揭开它们是如何影响转录输出的。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、生化和计算方法定义新的机制 它是一种知之甚少的组蛋白H3 PTM，H3苏氨酸45磷酸化(PH3T45)，传递细胞信号 从上游激酶到影响基因表达，并利用这种方法来描绘新的致病机制 组蛋白H3变异体调节表观基因组以改变细胞功能。我们的初步数据显示，H3T45 磷酸化状态(1)通过引导特定的H3K4修饰复合体来调节H3K4甲基化 染色质；(2)通过竞争性的组蛋白乙酰化/去乙酰化来干扰H3K36me3；(3)调节RNA 通过与剪接因子和RNA外体复合体的差异关联进行处理。我们将解剖 H3K4-甲基转移酶复合体在结构、功能和生物产量上的差异 PH3T45或未改性的H3T45。我们将描述PH3T45如何影响H3K36乙酰化动力学和 DNA修复背景下的H3K36甲基化。然后，我们将定义PH3T45如何管理 通过研究未修饰的H3T45和PH3T45在RNA加工过程中的作用来成熟RNA。最后，我们 已经确定了一系列与癌症相关的H3变种，在这些变种中，氨基酸被改变为赖氨酸，称为 “H3X to K”的变种。我们的初步数据表明，H3X到K变异体对近端H3PTM的调节失调 以独特的方式调节基因表达。我们将利用我们研究PH3T45的经验来机械地 定义H3X到K变异如何重新编程表观基因组以产生转录和功能细胞 改变。这项研究将解决组蛋白PTM模式和H3变异体的调节和作用 表达，这将告诉我们关于H3PTM和变异表达是如何导致人类疾病的观点。

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.