Abstract 5160: Development of a non-radioactive method to determine Protein-Methyl-Transferase activity in-vitro

• Published in: Cancer research (Chicago, Ill.) Ill.), 2014-10, Vol.74 (19_Supplement), p.5160-5160
• Main Authors: Mueller, Daniel, Beisenherz-Huss, Christian, Heidemann-Dinger, Carolin, Ketterer, Constance, Krämer, Diane, Kubbutat, Michael H.G.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2014-5160

The epigenetic regulation of gene expression has been of increasing interest in the last couple of years. It has been shown that methylation of DNA may pass information epigenetically even from one cellular generation to the next without changing the DNA sequence. Epigenetic information may also be transferred by acetylation, methylation and phosphorylation of DNA binding proteins, e.g. transcription factors and histones. The latter constitute the core of the nucleosomal particles, which are essential for structuring the DNA into chromatin. Histones are methylated on specific lysine or arginine residues by a family of enzymes called HMTs (Histone Methyl Transferases). It leads to either compacting or loosening of the chromatin structure which influences the transcriptional activity of the DNA bound to these histones. Histones in each nucleosomal particle may be differentially methylated and the exact information coded into such methylation patterns is not yet well understood. Overexpression of several HMTs, however, has been found in a number of pathological conditions including cancer. Therefore HMTs are a promising target class for pharmacological intervention in such diseases. To start a compound development process targeting specific HMTs, but also to enable profiling against many different HMTs, robust in-vitro assays are required that allow the generation of scientifically valid results, are preferably automatable, economically feasible and safe, and are readily transferable to all HMTs. Current methods to detect HMT activity monitor the transfer of the methyl-group from the co-substrate SAM (S-adenosyl-methionine) onto the protein substrate, either directly by tracing the 3H-labelled methyl-group from SAM to a substrate, or indirectly by using methylation site specific antibodies. Alternatively the reaction co-product SAH (S-adenosyl-homocysteine) may be detected directly by antibodies or indirectly via conversion of the SAH into other compounds which may be traced by various methods. While the use of 3H is often limited due to safety reasons, antibody based detection is frequently limited by antibody specificity, is costly and, in the case of site specific antibodies, results may only to a limited degree be compared when different sites are methylated by different HMTs. We have therefore developed an universal, non radioactive, non-antibody based in-vitro assay which detects HMT activity by its co-product SAH that is converted by a two-step, enzyma