An Integrated Approach for Combinatorial Readout of Dual Histone Modifications by Epigenetic Tandem Domains

Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However, it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (trimethylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by isothermal titration calorimetry and proximity ligation assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus, a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein interactions was successfully developed and can be adapted for broad biomedical applications.