Identification of dual histone modification-binding protein interaction by combining mass spectrometry and isothermal titration calorimetric analysis

Pu Chen; Zhenchang Guo; Cong Chen; Shanshan Tian; Xue Bai; Guijin Zhai; Zhenyi Ma; Huiyuan Wu; Kai Zhang

doi:10.1016/j.jare.2019.11.003

Identification of dual histone modification-binding protein interaction by combining mass spectrometry and isothermal titration calorimetric analysis

J Adv Res. 2019 Nov 13:22:35-46. doi: 10.1016/j.jare.2019.11.003. eCollection 2020 Mar.

Authors

Pu Chen¹, Zhenchang Guo¹, Cong Chen¹, Shanshan Tian¹, Xue Bai¹, Guijin Zhai¹, Zhenyi Ma¹, Huiyuan Wu², Kai Zhang¹

Affiliations

¹ 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China.
² School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China.

Abstract

Histone posttranslational modifications (HPTMs) play important roles in eukaryotic transcriptional regulation. Recently, it has been suggested that combinatorial modification codes that comprise two or more HPTMs can recruit readers of HPTMs, performing complex regulation of gene expression. However, the characterization of the multiplex interactions remains challenging, especially for the molecular network of histone PTMs, readers and binding complexes. Here, we developed an integrated method that combines a peptide library, affinity enrichment, mass spectrometry (MS) and bioinformatics analysis for the identification of the interaction between HPTMs and their binding proteins. Five tandem-domain-reader proteins (BPTF, CBP, TAF1, TRIM24 and TRIM33) were designed and prepared as the enriched probes, and a group of histone peptides with multiple PTMs were synthesized as the target peptide library. First, the domain probes were used to pull down the PTM peptides from the library, and then the resulting product was characterized by MS. The binding interactions between PTM peptides and domains were further validated and measured by isothermal titration calorimetry analysis (ITC). Meanwhile, the binding proteins were enriched by domain probes and identified by HPLC-MS/MS. The interaction network of histone PTMs-readers-binding complexes was finally analyzed via informatics tools. Our results showed that the integrated approach combining MS analysis with ITC assay enables us to understand the interaction between the combinatorial HPTMs and reading domains. The identified network of "HPTMs-reader proteins-binding complexes" provided potential clues to reveal HPTM functions and their regulatory mechanisms.

Keywords: Binding proteins; Histone; Posttranslational modification; Protein-protein interaction; Reader proteins.