Preparation of the ubiquitination-triggered active form of SETDB1 in Escherichia coli for biochemical and structural analyses

Abstract

Abstract Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1.