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In vitro studies of histone tail cross-talk effects on histone demethylases. Deciphering epigenetic marks on the molecular level. Brian Lohse*, Jan Kristensen, Charlotte Helgstrand, Ulrike Leurs, Paul A. C. Cloos, Rasmus P. Clausen, Jesper L. Kristensen. University of Copenhagen, Universitetsparken 2, 2100 København Ø, Denmark. Introduction Highlights DNA is wound tightly around histone complexes (see figure 1) and from the histone complexes long peptides are protruding perpendicularly, called histone tails (indicated by arrow on figure 1). We did the following observations for the enzymatic activity of the lysine demethylases: PTM cross-talk effects in vitro for Full length JMJD2C JMJD2A, JMJD2C and PHF8 1) H3K9(Me)3 > H3K9(Me)2 > H3K9(Me)1 2) H3K9(Me)3 < H3K9(Me)3K14(Ac) 3) H3K4(Me)3K9(Me)3 > H3K9(Me)3 4) H3K9(Me)3T11(ph) = No activity Enzyme Kinetics The correlation between the catalytic activity of 2C (see fig. 3), 2A & PHF8 (see fig. 4) as a function of peptide length on di- and trimethylated substrates on H3K9: Figure 1: DNA wound around a histone complex. Arrow indicates a histone tail. 0,9 JMJD2C (Ac)1: (Me)3 0,8 JMJD2C (Ac)1: (Me)2 0,7 JMJD2C (Ac)0: (Me)3 0,6 kcat (min-1) These histone tails are constantly modified by a range of enzymes adding and removing groups, through chemical modifications primarily: phosphorylations, acetylations and methylations. In this study we focus on histone 3 (H3), it’s tail modifications (see figure 2) and compare three demethylating enzymes within the Jumonji family. 0,5 0,4 0,3 0,2 Figure 5: kcat / Km for Full Length JMJD2C without and with K4(Me)3 Showing a change in kcat / Km 0,1 0 0 5 10 15 20 25 30 35 40 Number of amino acids in the peptides Figure 2: Histone 3 tail and it’s known modification sites. Figure 3: kcat for JMJD2C vs. length of substrate. (Ac)1 means K14 is acetylated on (εN). (Me)3 means K9 is trimethylated and (Me)2 dimethylated both on (εN). Purpose of the investigation 1. To try and shead light on the impact of the chemical modifications, through histone tail substrate modification and mimicking. 2. To truncate the natural substrate (H3), to the shortest peptide possible still giving rise to lysine demethylation. 3. Is it possible to observe cross-talk effects in vitro by monitoring the effect on the histone lysine demethylases, upon presenting more than one chemical modification on the same substrate e.g. H3K9(Me)3-T11(phos). 45 Conclusion We have shown that it is possible to study cross-talk effects on histone modifying enzymes by histone tail mimicking. Through peptide synthesis, specific combinations can be synthesized and the effects studied on one enzyme at the time, providing an important tool to gain information on the impact of chemical modifications. We have used these results to find specific substrate based inhibitors, for candidates as anticancer drugs. References Lohse et al. Angew. Chem. Int. Ed. 2011, 50, 9100. Figure 4: kcat for JMJD2A and PHF8 vs. length of substrate. (Ac)1 means K14 is acetylated on (εN). (Me)3 means K9 is trimethylated and (Me)2 dimethylated both on (εN). Lohse et al. Bioorg. Med. Chem. 2011, 19, 3625. *Contact: [email protected]