It’s All About “Me”: Making and Assaying Active Histone Methyltransferases
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Histone Methyltransferases (HMTs) are emerging as new targets for drug discovery at a time when our knowledge of how these enzymes work in the cell is far from complete. In vivo,histone methyltransferases (HMTs) target specific histone lysine or arginine residues at particular locations in chromatin. However, isolated HMTs often have low activity in vitro. While activity can sometimes be enhanced by addition of their in vivo binding partners, these are not always known. An added difficulty is that few known HMT inhibitors are potent or selective enough to act as assay validation controls. In this presentation, we will describe efforts to improve the quality and activity of recombinant HMTs, to develop precise biochemical assays, and to discover tool compounds for this new area of biology.
This webinar considers in somewhat greater detail case studies of several particular HMTs, focusing, for example, on our efforts to optimize the substrate and assay conditions for and characterize the kinetics of the histone H3 lysine-36 (H3K36) methyltransferase NSD2 (WHSC1, MMSET). Overexpression of NSD2 due to the t(4;14) translocation in multiple myelomas, can help drive oncogenesis. This effect may be phenocopied, particularly in certain leukemias and lymphomas, by what are presumed to be hyperactivating NSD2 mutations. We have investigated the effects of two such mutations, E1099K and T1150A, using full-length, insect cell-expressed, wild-type and mutant enzymes to perform in vitro kinetic studies with nucleosomal substrates. We find that while the E1099K enhancement of activity relative to wild-type is marginal under our standard methyltransferase assay conditions, it can be increased depending on pH, salt concentration and other factors. The T1150A mutation, however, robustly enhances activity across a broad range of conditions.
Three Key Takeaways from this webinar:
1 Learn about the tools and techniques used to characterize the activity of histone methyltransferases, enzymes that catalyse the formation of an important group of epigenetic post-translational modifications (lysine mono-, di-, and tri-methylation, arginine mono- and di-methylation).
2: Identify and explore some of the common challenges faced when developing biochemical histone methyltransferase assays for purposes of both basic enzymological and drug discovery research.
3: Learn in more detail about the properties of several HMTs that are emerging as targets of interest for drug discovery.
Challenges to be Addressed:
Preparations of purified recombinantHMTs can have quite low activity in vitro, often with turnover numbers of <1 h-1. However, biochemical assay conditions for purposes of high-throughput compound screening and other drug discovery applications are ideally performed at low enzyme concentrations, if possible nanomolar or lower. Thus, optimization of HMT protein preparations, substrate preparations and assay conditions can be critically important in determining the feasibility of targeting particular HMTs for inhibitor discovery and drug development.
Solutions of Focus
This webinar provides a comprehensive overview and some specific strategies to solve problems commonly encountered when assaying HMTs. Various approaches for enhancing the activity of purified recombinant HMT proteins will be discussed, including the preparation of multi-protein enzyme complexes, types of substrates to consider and the optimization assay buffer components.
Who should view
All scientists who are working in epigenetic fields from both industry and academic labs - Directors, Senior Scientists, research associates, scientists.
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Dr. Howitz received his Ph.D. in Biochemistry from Cornell University. After postdoctoral research at the University of Pennsylvania, he joined Biomol Research Laboratories, where he established Biomol’s recombinant enzymes product line and developed assay kits for a variety of enzymes, including the popular Fluor de Lys HDAC assays.
In 2010 Dr. Howitz joined Reaction Biology Corporation as its Director of Epigenetics and established its proteins group, which is currently focused on histone methyltransferases and demethylases and epigenetic reader domains.