Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes

Abstract

The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1’s binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2. To better understand why such a trivalent additive effect is lacking, we solved the cryo-EM structure (3.38 Å) of the complex of 53BP1 with the H2AXK15ub/S139ph_H4K20me2 nucleosome, which showed that H2AXS139 phosphorylation distorts the interaction interface between ubiquitin and 53BP1’s UDR motif. Our study revealed that there is redundancy in the interplay of multiple histone PTMs, which may be useful for controlling the dynamic distribution of effector proteins onto nucleosomes bearing different histone variants and PTMs in a time-dependent fashion, through specific cellular biochemical events.

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

“…Taken together, our results establish a conceptually new family of chemical tools for time-resolved studies on protein ubiquitination in live cells and further exemplify the utility of chemically synthesized customized probes that are more readily synthesizable and are of different structural designs for the study of Ub modifications. − It should be mentioned that although the current photocaging group, Nbg, requires 365 nm of UV light and may cause cytotoxicity in some experiments, our probe can be easily extended to use more benign photosensitive groups that would meet the requirements for specific biological conditions.…”

Synthesis of ubiquitinated proteins for biochemical and functional analysis

“…This synthetic target was prepared by expressed protein hydrazinolysis and auxiliary-mediated protein ligation. 100 The Ub was introduced using the 1,3-dibromoacetone strategy (Fig. 10).…”

The expedient, CAET-assisted synthesis of dual-monoubiquitinated histone H3 enables evaluation of its interaction with DNMT1

“…1,2 Our understanding of these processes necessitates the use of homogenously modied histones to assign functional consequences to site-specic ubiquitin marks. [3][4][5][6][7][8][9][10][11] In the past decade, site-selective conjugation chemistry using recombinant proteins as starting materials has been an attractive technology for making ubiquitinated histones, thereby facilitating the functional and mechanistic studies of histone ubiquitination in a chemically dened manner. 3,8,[12][13][14][15][16][17][18] For instance (Fig.…”

“…[3][4][5][6][7][8][9][10][11] In the past decade, site-selective conjugation chemistry using recombinant proteins as starting materials has been an attractive technology for making ubiquitinated histones, thereby facilitating the functional and mechanistic studies of histone ubiquitination in a chemically dened manner. 3,8,[12][13][14][15][16][17][18] For instance (Fig. 1a), disulde-exchange and a-halogen ketone-directed methods have been developed for histone site-specic monoubiquitination, [12][13][14] and the resulting histones were widely used in biochemical and structural studies.…”

“…1a), disulde-exchange and a-halogen ketone-directed methods have been developed for histone site-specic monoubiquitination, [12][13][14] and the resulting histones were widely used in biochemical and structural studies. 3,[19][20][21][22] In a recent study, hydrazide mimics of acylated histones were synthesized to evaluate their effects on nucleosome dynamics and assess deubiquitinase action. 17 The functionalization of dehydroalanine, a method that allows the installation of various natural PTMs and unnatural labels, [23][24][25][26][27] also shows potential for the chemical synthesis of homogenously ubiquitinated histones.…”