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

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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.