Generation of nonhydrolyzable ubiquitin-histone mimics

Abstract

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.

Keywords: Effector proteins; Histone modification; Ubiquitin.

Figure 1

Synthesis and purification of ubiquitinated histone mimics. (A) Schematic comparing native ubH2A and the crosslinked mimic. In the mimic, Gly76 of Ub and Lys-119 of H2A were mutated to cysteines. Crosslinking with 1,3-dicholoroacetone produces a dithioether linkage that is one C-C longer than an isopeptide linkage, contains an additional carboxylate, and is resistant to cleavage by DUBs (origninally published in [22]). (B) Pilot-scale crosslinking reactions were done by varying the ratio of Ub and histones. Products were separated by SDS-PAGE and visualized by Coomassie blue staining. (C) Crosslinked products were purified using Ni-NTA agarose. Eluates (containing Ub, Ub*Ub, and Ub*histones) were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) HPLC chromatogram of purification of ub*H2A/H2B dimers. Dimers assembled as described in section 2.4 were injected on a Superdex 75 column. Absorbance at 280 nm was monitored over time and eluates were visualized by SDS-PAGE and Coomassie blue staining. ub*H2A/H2B eluted at ~10.4 ml. H2B, Ub*Ub, and Ub eluted at approximately 7.9, 13.2, and 15.1 ml, respectively. (E) 1 μg of purified, unmodified or Ub*histone-containing dimers and octamers were analyzed by SDS-PAGE and Coomassie blue staining. (F) Purified histone octamers containing unmodified histones, ub*H2A or ub*H2B mimics, were assembled into mononucleosomes with 183mer DNA containing a centrally-localized 601 sequence. Assembly was done by salt dilution [16] and the octamer:DNA ratio was titrated in order to achieve the optimal condition.

Figure 2

A silver-stained gel of proteins bound to histone dimers containing unmodified histones, ub*H2A or ub*H2B mimics. Pulldown performed with Flag-Ub serves as a control.