Exploring the functional role of tubulin methylation and its regulation by mes-4/NSD in C. elegans

摘要 微管细胞骨架在细胞中起着许多关键功能，其功能障碍与细胞数量过多有关。 从癌症到自闭症等神经发育障碍的疾病。翻译后修饰(PTM) 微管的微管蛋白亚基是微管结构和功能的关键调节因子。类似 对于“组蛋白密码”，“微管密码”假说假设微管功能是通过 特定微管蛋白异构体和PTMS的掺入。众所周知，甲基化是组蛋白上的一种常见的PTM， 然而，微管蛋白甲基化的功能以及“读、写和擦除”这一点的酶机制 微管上的PTM在很大程度上还没有被探索。我们已经确定了组蛋白的新角色- 甲基转移酶NSD3是一种微管蛋白甲基转移酶，它使-微管蛋白在赖氨酸96和112( K96me2和K112me2)。我正在探索这些新的甲基标记在体内对-微管蛋白利用的作用 模式生物秀丽线虫。我现在发现蠕虫中的NSD3同源基因MES-4有一个体细胞 在神经元中的作用，导致我假设在K96和K112处MES-4的缺失取消了α-微管蛋白甲基化， 导致神经元细胞骨架的组织和功能缺陷。要在Aim1中验证这一假设，我 将利用缺乏甲基的敲打蛋白微管蛋白突变来探索K96me2和K112me2在蠕虫中的功能 为了了解这些位点甲基化缺失如何影响微管结构(Aim1.1)和动力学 (Aim1.2)。在目标2中，我将使用成像进一步确定MES-4的缺失是否会导致K96me2和/或K112me2的缺失 和生化技术(Aim2.1-2.2)。利用功能丧失探索这些甲基标记的作用 这些方法将得到MES-4功能研究(目标2.3)的补充。许多人类癌症是由 通过过度/结构性激活NSD3的突变。我会产生一个携带相同基因的MES-4突变蠕虫 在人类癌症中发现的线虫相应(保守)位置的突变，以询问MES-4是否过度活跃 导致细胞骨架和功能缺陷。我的博士论文将深入研究一个新的 关于表观遗传机制如何调节细胞骨架的观点，对 了解与细胞骨架缺陷有关的许多疾病。

ABSTRACT The microtubule cytoskeleton serves many critical functions in the cell, and its dysfunction is linked to a plethora of diseases from cancer to neurodevelopmental disorders such as autism. Post-translational modifications (PTM) of the tubulin subunits of microtubules are key regulators of both structure and function of microtubules. Similar to the “Histone Code”, the “Tubulin Code” hypothesis posits microtubule function is tuned through the incorporation of specific tubulin isoforms and PTMs. Methylation is well known as a common PTM on histones, however, the function of tubulin methylation and the enzymatic machinery that “reads, writes and erases” this PTM on microtubules has been largely unexplored. We have identified a new role for the histone- methyltransferase NSD3 as a tubulin methyltransferase that di-methylates -tubulin at lysines 96 and 112 (the K96me2 and K112me2 marks). I am exploring the in vivo role of these new methyl marks on -tubulin utilizing the model organism C. elegans. I have now discovered the NSD3 orthologue in the worm, mes-4, has a somatic role in neurons, leading me to hypothesize loss of mes-4 abrogates α-tubulin methylation at K96 and K112, resulting in defects in organization and function of the neuronal cytoskeleton. To test this hypothesis in Aim1 I will explore the function of K96me2 and K112me2 in the worm utilizing methyl-deficient knockin tubulin mutations to understand how lack of methylation at these sites impacts microtubule structure (Aim1.1) and dynamics (Aim1.2). In Aim 2, I will further determine if loss of mes-4 causes loss of K96me2 and/or K112me2 using imaging and biochemical techniques (Aim2.1-2.2). Studies exploring the role of these methyl marks using loss of function approaches will be complemented by mes-4 gain of function studies (Aim 2.3). Many human cancers are driven by mutations that over/constitutively activate NSD3. I will generate a mes-4 mutant worm carrying the same mutation at the corresponding (conserved) site in C. elegans seen in human cancers, to ask if mes-4 hyperactivity induces cytoskeletal and functional deficits. My doctoral dissertation will thoroughly investigate a new perspective on how epigenetic machinery regulates the cytoskeleton, with far reaching implications for understanding the many diseases linked to cytoskeletal defects.