Regulation and recognition of H3K79me

项目总结/摘要 核小体组蛋白的动态翻译后修饰模式调节核小体的局部和 通过募集专门的效应蛋白来实现全局真核基因表达。单甲基化、二甲基化和三甲基化 组蛋白H3赖氨酸79（H3 K79 me 1/2/3）是活跃转录基因的标志， 基本的生物过程。重要的是，H3 K79的异常高甲基化在混合型中被发现。 谱系白血病1-重排（MLL-r）白血病，对治疗有抵抗力。新的有效治疗方法， 这些白血病是迫切需要的，但H3 K79 me调控的潜在机制， 认识不足。Dot 1 L是负责建立H3 K79 me的唯一甲基转移酶。 它由组蛋白H2 B（H2 Bub）的单泛素化刺激。在细胞中，发现Dot 1 L与伴侣复合 已知蛋白AF 9和AF 10上调H3 K79 me水平。然而，人们对 它们对Dot 1 L结合亲和力和对H2 Bub修饰的核小体的活性的影响。在目标1中，我将比较 Dot 1 L + AF 9和Dot 1 L + AF 10对含H2 Bub的核小体的结合亲和力和活性， 使用生化技术进行H3 K79 me 1/2/3修饰。以指导未来基于结构的白血病 此外，我还将确定H2 Bub核小体上Dot 1 L + AF 9 + AF 10的冷冻电镜结构。 已知H3 K79 me 2/3调节基本的生物学过程，但没有H3 K79 me效应蛋白具有 被识别。H3 K79位于核小体的球状核心内，可能需要使用 核小体底物作为诱饵在下拉实验中鉴定H3 K79 me结合蛋白。但没有 下拉研究已经使用H3 K79 me修饰的核小体来鉴定结合蛋白。在目标2中，我将 使用H3 K79 me 2/3修饰的核小体作为诱饵在蛋白质下拉中鉴定H3 K79 me结合蛋白， 哺乳动物核提取物。我会用基于质谱的蛋白质组学来识别潜在的命中， 使用生物化学技术验证它们的特异性H3 K79 me结合。最后，研究其作用机理 通过确定H3 K79 me结合蛋白的cryo-EM结构， 核小体目标1和目标2中这些拟议实验的成果将成为指导 设计靶向Dot 1 L伴侣蛋白的基于药物的白血病疗法，并将揭示H3 K79甲基 标记被效应蛋白识别以调节基因表达。

Project Summary/Abstract Patterns of dynamic post-translational modifications on histone proteins of nucleosomes regulate local and global eukaryotic gene expression by recruiting specialized effector proteins. Mono-, di-, and tri- methylation of histone H3 lysine 79 (H3K79me1/2/3) is a hallmark of actively transcribed genes and is central to many fundamental biological processes. Importantly, aberrant hypermethylation of H3K79 is found in the Mixed Lineage Leukemia 1-rearranged (MLL-r) leukemias which are resistant to treatment. New potent treatments for these leukemias are desperately needed, but the underlying mechanisms of H3K79me regulation and recognition are poorly understood. Dot1L is the sole methyltransferase responsible for establishing H3K79me. It is stimulated by mono-ubiquitylation of histone H2B (H2Bub). In cells, Dot1L is found in complex with partner proteins AF9 and AF10, which are known to upregulate H3K79me levels. However, very little is known about their effects on Dot1L binding affinity and activity on H2Bub-modified nucleosomes. In Aim 1, I will compare the binding affinity and activity of Dot1L + AF9 and Dot1L + AF10 on nucleosomes containing H2Bub and H3K79me1/2/3 modifications using biochemical techniques. To guide future structure-based leukemia therapies, I will also determine the cryo-EM structure of Dot1L + AF9 + AF10 on H2Bub nucleosome. H3K79me2/3 is known to regulate fundamental biological processes, yet no H3K79me effector proteins have been identified. H3K79 is located within the globular core of the nucleosome and may require the use of nucleosomal substrates as bait in pulldown experiments to identify H3K79me binding proteins. However, no pulldown studies have used H3K79me-modified nucleosomes to identify binding proteins. In Aim 2, I will identify H3K79me binding proteins using H3K79me2/3-modified nucleosomes as bait in protein pulldowns from mammalian nuclear extract. I will identify potential hits using mass-spectrometry based proteomics and validate them for specific H3K79me binding using a biochemical technique. Finally, I will study the mechanism of H3K79me recognition by determining a cryo-EM structure of a high-affinity H3K79me binding protein on nucleosome. The outcomes from these proposed experiments in Aims 1 and 2 will form a basis for guiding the design of drug-based leukemia therapies targeting Dot1L partner proteins and will reveal how H3K79 methyl marks are recognized by effector proteins to modulate gene expression.