Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz’s auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5–6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond.

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.

Four diubiquitinated H2A analogues were newly synthesized and used to reveal the plasticity of ubiquitin chains in 53BP1 recognition.