EXPANDING THE CHROMATIN TOOLBOX: EMPLOYING DESIGNER CHROMATIN SUBSTRATES TO CHARACTERIZE HISTONE PTMS AND MUTATIONS

Abstract

Chemical methods for studying biology are invaluable tools for elucidating biochemical mechanisms that govern the fundamental processes of life. In this thesis, we employed and expanded the existing chemical biology toolkit to study both TGM2-mediated monoaminylation of histones and nucleosome asymmetry in chromatin. Using a biorthogonal labeling strategy, we successfully demonstrated that histones are subject to serotonylation at H3Q5. A detailed analysis revealed that H3Q5ser exists in combination with H3K4me3 to mark regions of active chromatin. We next used existing methods to produce a diverse set of chemically defined nucleosomal substrates to identify chromatin properties that govern the substrate selectivity of TGM2. We discovered that, in addition to H3Q5, H3Q19 and H2A.ZQ123 and Q124 are glutaminyl substrates for TGM2 in vitro. Modification of these residues occurs in a sequence-independent manner, with the primary determining factor for substrate selectivity being steric accessibility. Due to the promiscuity of TGM2, we wondered if we could develop new chemical methods to probe substrates of serotonylation proteome-wide. Leveraging the electron rich nature of the 5-hydroxy indole ring, we developed a chemoselective probe for serotonin. Finally, we expanded the existing chromatin toolkit to provide greater access to asymmetric chromatin substrates. We report application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes. This genetically encoded covalent tethering system serves as an internal chaperone, maintained through the assembly process, affording traceless asymmetric nucleosomes following proteolytic removal of the tethers. We used this system to interrogate the effects of asymmetry on chromatin remodeling, nucleosome stability, and histone PTM removal.