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

Abstract

Site-selective conjugation chemistry has proved effective to synthesize homogenously ubiquitinated histones. Recently, a powerful strategy using 2-((2-chloroethyl) amino) ethane-1-thiol (CAET) as a bifunctional handle was developed to generate chemically stable...

Efficient Chemical Synthesis of Multi-Monoubiquitylated and Diubiquitylated Histones by the α-Halogen Ketone-Mediated Strategy

“…The recent cell biology findings show that histones can be simultaneously monoubiquitylated at multiple lysine residues. A representative example is H3 ubiquitylation at the adjacent lysines (positions 14, 18, and 23) catalyzed by the E3 ligase Uhrf1, which is essential for the functions of the maintenance DNA methyltransferase DNMT1. , We and others recently revealed that H3 dual monoubiquitylation at K18 and K23 can directly interact with and activate DNMT1, ,, but the consequences of other H3 ubiquitylation patterns on this process are still unclear. For example, the binding affinity of DNMT1 with other dual-monoubiquitylated H3 proteins (K14- and K18- or K14- and K23-dual-monoubiquitylated one) or triple-monoubiquitylated H3 has not been evaluated yet, which is highly dependent on the access of site-specifically ubiquitylated H3 proteins.…”

“…Notably, the increase in potential byproducts and steric hindrance might make the synthesis of multi-monoubiquitylated histones more challenging, and their synthesis has not been reported yet. Actually, our recently reported method that was developed to generate the H3K18UbK23Ub analogue was difficult in the preparation of H3Ub3 …”

“…With a series of single-, dual-, and triple-monoubiquitylated H3 proteins in hand, we then explored their effects on the binding of DNMT1 to nucleosomes. It should be noted that although the interaction of DNMT1 with H3K18 C UbK23 C Ub has been well characterized, , the effects of H3Ub3, H3K14 C UbK18 C Ub, or H3K14 C UbK23 C Ub on the binding of DNMT1 to nucleosomes have not been studied. These H3 proteins, together with recombinant H2A, H2B, and H4, were reconstituted into histone octamers, which were then assembled to nucleosome core particles (NCPs) using 147 bp of Widom 601 targeting sequence under the standard condition , (Figure d,e and Figure S7a).…”

“…Actually, our recently reported method that was developed to generate the H3K18UbK23Ub analogue was difficult in the preparation of H3Ub3. 40 Similar to the preparation of mutants 2 and 7, the H3 mutant containing three K-to-C mutations at positions 14, 18, and 23 (H3K14CK18CK23C, 14) was recombinantly expressed in E. coli (Figure S4g). The conjugation of three ubiquitin monomers to H3 was performed by mixing proteins 3 and 14 in boric acid buffer at a molar ratio of 6:1.…”

“…Histone ubiquitylation, in which ubiquitin (Ub) is installed onto the lysine residue of histones via an isopeptide bond, is a critical post-translational modification (PTM) for various chromatin-related processes, such as DNA replication, transcription, and repair. − These very different outcomes may result from the diversity of histone ubiquitylation in structures, such as monoubiquitylation bearing a Ub monomer on one lysine, multi-monoubiquitylation in which Ub monomers are conjugated to histones on multiple residues, and polyubiquitylation in which Ub chains are installed onto histones. − The decoding of ubiquitylation-dependent regulation requires the use of precisely ubiquitylated histones to allocate their functions and reveal the underlying molecular mechanisms. − Therefore, remarkable endeavors have been focused on the chemical preparation of those histones. − Total chemical synthesis − and semisynthesis − enable the generation of ubiquitylated histones with a native linkage, while site-specific conjugation chemistries − allow us to produce ubiquitylated histone analogues from recombinant materials. Among these methods, the α-halogen ketone-mediated strategy (Scheme a) has gained attractive attention and been broadly applied by the biology community due to its practicality in preparing ubiquitylated histones and the resistance of the resulting linkages to deubiquitinases.…”