Regulation and recognition of H3K79me

项目摘要/摘要 核小体组蛋白的动态翻译后修饰模式调节局部和 通过招募特化效应蛋白进行全球真核基因表达。单甲基化、双甲基化和三甲基化 组蛋白H3赖氨酸79(H3K79me1/2/3)是活跃转录基因的标志，是许多基因的中心 基本的生物过程。重要的是，在混合病例中发现了H3K79的异常高甲基化 耐药的谱系白血病1-重排(MLL-r)白血病。新的有效治疗方法 这些白血病是迫切需要的，但H3K79me调节和转录的潜在机制 人们对其认可度知之甚少。DOT1L是唯一负责建立H3K79Me的甲基转移酶。 它是由组蛋白H_2B(H_2Bub)的单一泛素化所刺激的。在细胞中，DOT1L存在于与伴侣的复合体中 AF9和AF10蛋白，已知可以上调H3K79me的水平。然而，人们对此知之甚少。 它们对H_2Bub修饰的核小体上DOT1L结合亲和力和活性的影响。在目标1中，我将比较 DOT1L+AF9和DOT1L+AF10与含H_2Bub和H_2Bub的核小体的结合亲和力和活性 使用生化技术对H3K79me1/2/3进行修饰。指导未来基于结构的白血病 在治疗过程中，我还将确定DOT1L+AF9+AF10在H_2Bub核小体上的冷冻EM结构。 已知H3K79me2/3调节基本的生物学过程，但还没有H3K79me效应蛋白 已被确认身份。H3K79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H3K79me结合蛋白。然而，没有 下拉研究已经使用H3K79Me修饰的核小体来鉴定结合蛋白。在《目标2》中，我会 用H3K79me2/3修饰的核小体作为诱饵鉴定H3K79me结合蛋白 哺乳动物核提取液。我将使用基于蛋白质组学的质谱学识别潜在的命中结果 使用生化技术验证它们是否与特定的H3K79me结合。最后，我将研究一下这个机制 通过确定高亲和力H3K79me结合蛋白的冷冻EM结构来识别H3K79me 核小体。目标1和目标2中这些拟议实验的结果将成为指导 针对DOT1L伙伴蛋白的药物白血病治疗设计将揭示H3K79如何甲基化 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.