Protein Engineering Tools to Explore the Function of Protein Post-Translational Modifications for Chromatin and Microtubule Cytoskeletal Biology

Protein post-translational modifications (PTMs) play a crucial role in expanding the protein diversity and are one of the major mechanisms through which cells respond to ever changing environmental cues. The function of two of the most important cellular complexes, chromatin and microtubules, is influenced and very tightly regulated by underlying protein post-translational modifications. The focus of my thesis is to develop tools to investigate the role of protein PTMs in the context of chromatin and microtubules.

Eukaryotic DNA is organized in the form of chromatin whose basic unit is the nucleosome. The nucleosome is composed of 147 base pair of DNA wrapped around four core histones forming H2A, H2B, H3 and H4 forming an octamer. Each histone can be highly post-translationally modified, especially on their N-terminal tails protruding from the nucleosome particle. Histone post-translational modifications (PTMs) work combinatorially to establish chromatin states defined by specific gene expression status known as the Histone Code. Although, each nucleosome carries two copies of each histone, each copy in a single nucleosome can be differently modified resulting in PTM based nucleosome asymmetry. In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), histone H3 methylated at lysine 4(H3K4me3)- an activating histone PTM mark, coexists with H3K27me3- a repressive histone PTM mark, in asymmetric nucleosomes. In the first project, a general, modular and a traceless synthetic strategy to produce asymmetrically modified nucleosomes is described. Using these asymmetric nucleosomes, I show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27-specific lysine methyltransferase (KMT) Polycomb Repressive Complex 2 (PRC2) solely on the same histone tail. Whereas H3K27me3 stimulates PRC2 activity via a positive feedback mechanism across tails, thereby partially overriding the H3K4me3-mediated repressive effect.

Microtubules are the largest components of the eukaryotic cytoskeleton and play an import role in maintain cellular organization, intracellular and neuronal transport, cell motility and cell division. Microtubules are dynamically assembled from αβ-tubulin heterodimer, which is the basic repeating unit. The primary sequence and structure of tubulin proteins and microtubules are highly conserved in eukaryotic evolution. Despite this conservation, tubulin is subjected to heterogeneity that arises from differential expression of multiple tubulin isotypes and the vast repertoire of tubulin PTMs, predominantly on the unstructured C-terminal tubulin tails. Together, this gives rise to the Tubulin Code. Lack of access to uniformly modified tubulin presents a major difficulty in study of PTM function. Here, I have developed a method to link synthetic, modified tails to recombinant tubulin by use of a split intein based, protein trans-splicing (PTS) approach. I demonstrate this approach by linking a fluorescently modified tubulin tail to recombinant human tubulin, preparing semisynthetic tubulin dimers. Extending the method to native PTMs will open the door for an in-depth study of tubulin modification using a chemically defined system.