A Scalable Platform for Producing Recombinant Nucleosomes with Codified Histone Methyltransferase Substrate Preferences

Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1; also known as NSD2) is a SET domain-containing histone lysine methyltransferase. A chromosomal translocation occurs in 15-20% of multiple myeloma patients and is associated with increased production of WHSC1 and poor clinical prognosis. To define the substrate requirements of NSD2, we established a platform for the large-scale production of recombinant polynucleosomes, based on authentic human histone proteins, expressed in E. coli, and complexed with linearized DNA. A brief survey of methyltransferases whose substrate requirements are recorded in the literature yielded expected results, lending credence to the fitness of our approach. This platform was readily 'codified' with respect to both position and extent of methylation at histone 3 lysines 18 and 36 and led to the conclusion that the most readily discernible activity of NSD2 in contact with a nucleosome substrate is dimethylation of histone 3 lysine 36. We further explored reaction mechanism, and conclude a processive, rather than distributive mechanism best describes the interaction of NSD2 with intact nucleosome substrates. The methods developed feature scale and flexibility and are suited to thorough pharmaceutical-scale drug discovery campaigns.