Generation of nonhydrolyzable ubiquitin-histone mimics.

Histone proteins undergo various types of post-translational modifications (PTMs) to regulate dynamic processes in the cell, including replication, transcription and DNA damage repair. One type of histone PTM is the attachment of a small protein, ubiquitin (Ub). In eukaryotic organisms, a single Ub is attached to specific lysine residues of histones H2A and H2B in a modification that, unlike many other forms of ubiquitination in the cell, does not signal degradation. Instead, both attachment and removal of Ub to these histones has been shown to affect gene transcription, pre-mRNA splicing, and DNA damage repair, but the mechanisms by which histone ubiquitination governs these processes are not well understood. In an effort to identify "readers" of Ub-histones, we developed a straightforward crosslinking strategy to generate nonhydrolyzable Ub-histone mimics. These mimics were assembled into Ub-histone-containing dimers or nucleosomes. We demonstrate that they can be used in pulldown assays to identify proteins that differentiate unmodified and ubiquitinated histones.