SYSTEMATIC ANALYSIS OF HISTONE H4 LYSINE 20 MODIFICATION FUNCTION IN GENE REGULATION, CELL PROLIFERATION, AND ANIMAL DEVELOPMENT

Histone post-translational modifications (PTMs) regulate processes necessary for gene regulation, cell proliferation, and animal development by modulating DNA accessibility and the recruitment of trans-acting factors. Therefore, enzymes that deposit histone PTMs (writers) and the modifications they deposit are crucial regulators of cell proliferation. Set8KMT5A is a writer responsible for mono-methylating the twentieth residue of histone H4 (H4K20me1). H4K20me1 is involved in numerous nuclear processes, but its function has largely been interpreted through manipulating the function of Set8. However, Set8 has several identified non-histone substrates that have essential roles in gene regulation and cell proliferation, such as p53 and PCNA, and proposed non-catalytic functions. Thus, a significant knowledge gap in the field is which functions of Set8 are attributable to H4K20me1 rather than non-histone substrates or non-catalytic interactions. Using Drosophila melanogaster as a model organism, this work utilizes a combination of genetic, genomic, biochemical, and molecular techniques to perform a systematic analysis of an allelic series of mutants involved in the H4K20 modification regulatory network. This work separates functions of H4K20 modification from pleiotropic functions of the H4K20me1 writer Set8 mono-methyltransferase and H4K20 reader L(3)mbt, and introduces a novel histone replacement platform that improves and expands upon current platforms. We find that modification of H4K20 is not essential for completion of development nor cell proliferation, and we implicate Set8 as a master regulator of genome function independent of its catalytic activity. Further, we demonstrate that L(3)mbt requires H4K20 modification for a portion of its chromatin occupancy. Together, these data present novel findings that would not have been possible using other experimental systems and have wide-ranging implications in the treatment of diseases such as cancer.