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Genetic Engineering & Biotechnology News | GENengnews.com | SEPTEMBER 1, 2016 | 17 Additionally, these types of assays are gener- ally less sensitive and require higher enzyme concentrations. Substrate Choices An additional challenge to developing these assays is the choice of substrate. The biological targets of the epigenetic enzymes within the cell are not free histones. Rather, histone targets are organized into nucleo- somes that are further packaged into high order structures within chromatin. The nu- cleosome consists of histone octamers made up of two copies each of histones H2A, H2B, H3, and H4, around which DNA is wound. In regions containing actively expressed or transcribed genes, chromatin exists in an open and accessible structure, while, in tran- scriptionally repressed regions, chromatin is more condensed. The complexity and structural variability of chromatin as the natural substrate repre- sents a unique drug discovery challenge for these classes of enzymes because it is difficult to reproduce the chromatin structure in vi- tro. Therefore, alternative substrates, such as short modified peptides that can effectively mimic the histone tail, are often used in high- throughput screens. However, peptides do not reproduce the complexity of the native biological substrate and do not always interact with epigenetic enzymes in a way that yields a productive reaction. Other types of substrates have been shown to perform better with specific enzymes and include 1) modified or unmodi- fied full-length histones, 2) histone octam- ers, 3) mononucleosomes, and 4) oligonu- cleosomes (Figure 1). Additionally, many of these substrates can be generated to include histone variants, such as H3.1 vs H3.3, or histone H3 con- taining cancer-associated mutations, such as lysine 27 to methionine (K27M), glycine 34 to arginine/valine (G34R/V) or lysine 36 to methionine (K36M) mutations that occur in some childhood cancers. Therefore, criti- cal decisions need to be made during assay development with regard to what substrate choice will perform best with its associated enzyme in the assay. Enzyme-Dependent Substrate Preferences To illustrate how substrate choice may influence assay results, we chose to exam- ine the activity of two histone methyltrans- ferases, PRC2 and SETD2, in the presence of either short histone H3 or histone H4 peptides, full-length recombinant histone H3, octamers and nucleosomes (Figure 2). EZH2, the catalytic component of the mul- tiprotein PRC2 complex, methylates histone H3 lysine 27, while SETD2 is a methyltrans- ferase that catalyzes the methylation of his- tone H3 lysine 36. The substrate preferences of the PRC2 and SETD2 methyltransferases were ex- amined using an assay that measures the production of S-Adenosyl-L-homocysteine (SAH) from the methyl group donor S-Ade- nosyl methionine (SAM), a cofactor used by all methyltransferases. Measurement of SAH production as a catalytic by-product enables an unbiased comparison of substrate prefer- ence since SAH production is a reflection of catalytic activity and is not affected by sub- strate differences. The data presented in Figure 2A shows that the preferred substrate for PRC2 is the mononucleosome. While full-length histone, assembled octamers, and peptide substrate are effective as substrates, they are less effi- cient substrates of the PRC2 complex, sug- gesting that PRC2 activity is regulated by nucleosomal environment in vivo so as to reduce the nonspecific activities on free his- tones prior to chromatin integration. The result of the SETD2 assay in Fig- ure 2B also shows that enzyme activity is highly dependent on substrate selection. For SETD2, the nucleosome is the only effective substrate. This result is expected, as SETD2 is known to require intact nucleosomes and bind to histone H4 in the context of the nucleosome while depositing a methyl group on histone H3 lysine 36. Conclusions The data presented above demonstrate that there are clear differences in epigen- etic enzyme activities in biochemical assays depending on which substrate is provided. Having insight into the substrate require- ments of an enzyme along with an under- standing of the strengths and limitations of the selected assay platform are essential for developing successful and robust assays for epigenetic drug discovery. www.moleculardevices.com | 800.635.5577 For Research Use Only. Not for use in diagnostic procedures. © 2016 Molecular Devices, LLC. All Rights Reserved. The trademarks mentioned herein are the property of Molecular Devices, LLC or their respective owners. Unleash your brilliance™ ZOOM Y X Optimize your complex assays with 3D imaging and analysis • Extract three dimensional data from diverse sample types, from cell monolayers to organoids • Refine object identification using advanced filtering and segmentation tools • Generate 3D measurements of complex samples, as well as the substructures within • Visualize raw data and segmentation in the 3D space moleculardevices.com/xyzoom Mary Anne Jelinek, Ph.D. (mjelinek@ activemotif.com), is senior research scientist at Active Motif. Website: www.activemotif.com. DRUG DISCOVERY Assay Tutorial