Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform.

Tobias Wagner,Teresa Burgahn,Manfred Jung,Joel Mcmillan,Daumantas Matulis,Lina Baranauskienė,Karin Schmidtkunz,Udo Oppermann,Yan Xiong,Jian Jin,Roland Schüle,Oleg Fedorov,Anne-Kathrin Schott,Holger Greschik

doi:10.1093/nar/gkw089

Abstract

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

Highlights

Gene expression is regulated in a hierarchical way by various control-mechanisms
Alpha technology is making use of colloidal bead suspensions consisting of donor- and acceptor beads which produce a chemoluminescent signal after excitation at a certain wavelength if they are brought into close proximity
Streptavidin-coated donor beads were used to capture a biotinylated peptide derived from the endogenous ligand of Spindlin[1], namely histone protein H3 trimethylated at position 4 (H3K4me[3]; amino acids 1–23)

Summary

Introduction

Gene expression is regulated in a hierarchical way by various control-mechanisms. DNA methylation and subsequent oxidation of methylated cytosine residues within DNA and a variety of specific post-translational modifications (PTMs) of histone proteins are the main contributors to epigenetic regulation processes. Covalent PTMs of histone proteins that are created by so-called ‘writer’ enzymes or removed by enzymes termed ‘erasers’ include acetylation, phosphorylation, ubiquitination, ribosylation, sumoylation and methylation (1) These modifications have an intrinsic effect on chromatin structure and activity. Acetylation of lysine residues within histones leads to a loss of the positive charge of the ⑀-ammonium group under physiological conditions which results in a weaker interaction of negatively charged DNA with the histone proteins. This eventually causes a more accessible chromatin state, referred to as euchromatin (2). Posttranslational methylation of lysine residues does not alter the charge of the amino acid side chain and there is only a subtle

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