Analysis of histone methyltransferases in Drosophila development

In this thesis, I describe research performed on three different SET domain proteins in Drosophila. Chapter 1 is an overview of SET domain proteins and how they are involved in the regulation of chromatin structure and transcriptional control.;Chapter 2 describes the SET8 protein which methylates histone H4 on lysine 20. Collaborators purified the enzyme from Hela cells and characterized its HMTase activity in vitro. I studied the Drosophila homolog to gain insight into the SET8 function in vivo, including genetic analysis of a lethal P element insertion into the 5' UTR and distribution of the methyl-H3K20 on polytene chromosomes in both wild type and mutant larvae.;Chapter 3 investigates the role of Mes-4 in Drosophila development. Expression analysis of the dMes-4 mRNA showed it was present throughout development; however the developmental requirement for dMes-4 was not resolved by the mutagenesis screen. While the specificity of the HMTase activity was not determined for dMES-4, it is suggested to be H3K36 based on the activity of the mammalian homolog.;Chapter 4 focuses on the ESC-E(Z) Polycomb group complex that is an H3K27 specific HMTase. The E(Z) SET domain protein was previously shown to require assembly into the complex with the non-catalytic subunits for robust activity. This chapter investigates the roles of the non-catalytic subunits in assembly and potentiating the E(Z) HMTase in vitro, additionally for ESC, in vivo. Analysis of the homologous worm MES2,3,6 complex is included and shows a similar reliance on the non-catalytic subunits for robust HMTase activity. An appendix includes analysis of the nucleosomal binding activities of Drosophila wild type and mutant complexes and the MES wild type and subcomplexes.