Role of novel histone modifications and variants in transcriptional regulation

项目摘要 核小体相关组蛋白的翻译后修饰（PTM），沿着组蛋白变体 掺入，影响转录能力和它们的失调已经在许多 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; 这些PTM的精确组合可以调节染色质结构和基因组组织， 基因表达的变化。尽管进行了广泛的努力来表征H3 PTM和H3变体，但它们的 机制和功能的相互作用，以及它们影响生物输出的能力，目前还不清楚如何 组蛋白和许多组蛋白PTM整合来自上游信号级联的信号以调节基因表达。 我实验室的首要目标是确定调节组蛋白PTM模式的机制， 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、生物化学和计算方法来定义新的机制， 其中一个知之甚少的组蛋白H3 PTM，H3苏氨酸45磷酸化（pH 3 T45）， 从上游激酶到影响基因表达，并利用这种方法来描述新的致病性 组蛋白H3变体使表观基因组失调以改变细胞功能。我们的初步数据表明，H3 T45 磷酸化状态（1）通过指导特定的H3 K4修饰复合物， 染色质;（2）通过竞争性组蛋白乙酰化/脱乙酰化破坏H3 K36 me 3;（3）调节RNA 通过与剪接因子和RNA外泌体复合物的差异缔合进行加工。我们将解剖 H3 K4-甲基转移酶复合物在结构、功能和生物输出方面的差异 pH 3 T45或未修饰的H3 T45。我们将描述pH 3 T45如何影响H3 K36乙酰化的动力学， DNA修复背景下的H3 K36甲基化。然后，我们将定义pH 3 T45如何控制 通过检查未修饰的H3 T45和pH 3 T45在整个RNA加工过程中的作用来检测成熟RNA。最后我们 已经鉴定了一系列癌症相关的H3变体，其中氨基酸被改变为赖氨酸，称为 “H3 X to K”变体。我们的初步数据表明，H3 X到K变异体失调近端H3 PTM 来独特地调节基因表达。我们将利用我们研究pH 3 T45的经验， 定义H3 X到K变体如何重编程表观基因组以产生转录和功能性细胞 变化本研究将探讨组蛋白PTM模式和H3变异体的调控和作用， 表达，这将告知我们如何H3 PTMs和变异体表达人类疾病的基础的看法。

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.