The
chemical synthesis of homogeneously ubiquitylated histones
is a powerful approach to decipher histone ubiquitylation-dependent
epigenetic regulation. Among the various methods, α-halogen
ketone-mediated conjugation chemistry has recently been an attractive
strategy to generate single-monoubiquitylated histones for biochemical
and structural studies. Herein, we report the use of this strategy
to prepare not only dual- and even triple-monoubiquitylated histones
but also diubiquitin-modified histones. We were surprised to find
that the synthetic efficiencies of multi-monoubiquitylated histones
were comparable to those of single-monoubiquitylated ones, suggesting
that this strategy is highly tolerant to the number of ubiquitin monomers
installed onto histones. The facile generation of a series of single-,
dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate
the influence of ubiquitylation patterns on the binding of DNA methyltransferase
1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific
conjugation chemistry to generate chemically defined histones for
epigenetic studies.