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Detection of JumonjiC Domain-Containing Histone Demethylase activities With a Homogeneous Bioluminescent Succinate Assay
EP25777
Poster Title: Detection of JumonjiC Domain-Containing Histone Demethylase activities With a Homogeneous Bioluminescent Succinate Assay
Submitted on 15 May 2017
Author(s): Hicham Zegzouti, Juliano Alves, and Said Goueli
Affiliations: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711
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Poster Information
Abstract: JumonjiC domain containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome, leading to either transcriptional repression or activation of target genes by counteracting the activities of histone lysine methyltransferases. Because of their implication in cancer, they have become validated drug targets. Therefore, assays for this enzyme subfamily are desirable in order to facilitate the identification of selective and potent inhibitors for drug discovery and as basic research tools. Since succinate is one of the products of the demethylation reaction catalyzed by these enzymes, an assay that detects succinate would be suitable for monitoring all JMJC activities as well as other succinate-forming enzymes (i.e.: dioxygenases). Thus, a bioluminescent and homogenous succinate detection assay for measuring JMJC activity was developed, and it is performed in a two-step format that relies on converting the succinate product to ATP, and then to light in a robust luciferase reaction. The light output is proportional to succinate concentration from low nM to 15μM, and the assay is highly sensitive and robust, two features that are highly desirable and essential for measuring the activity of the majority of JMJC demethylase subfamilies. Therefore, the succinate detection assay is a simple-to-use method that does not require antibodies or modified substrates. Examples of various applications of this succinate detection assay will be presented, including studies on specificity of different substrates by diverse JMJCs, as well as mode of action studies using specific inhibitors. The development of this succinate detection assay will make it possible to investigate a large number of JMJC demethylases and could have significant impact on diverse areas of Epigenetics research.Summary: JumonjiC domain-containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome,References: Report abuse »
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