Figure 2: Ubiquitylated H2B interacts with the N-terminal tail of H2A.
(a) SDS-PAGE analysis of the crosslinking reaction between biotinylated mononucleosomes containing H2BssUb* and a 40-fold molar excess of the catalytic domain of hDot1L. Reaction mixtures were purified by streptavidin pull-down before analysis—note, residual hDot1L remains non-covalently bound to mononucleosomes even after extensive washing. Addition of dithiothreitol, which cleaves the disulfide bond connecting Ub to H2B, led to the generation of a new species of lower molecular weight. Crosslinked species are indicated with blue arrows. (b) Western blot analysis of crosslinked species, with or without dithiothreitol treatment to complete the ubiquitin transfer step. Mononucleosomes containing H2BssUb* were UV irradiated and separated on a denaturing gel, followed by immunoblotting against ubiquitin (red) and H2A (green). The impurity (#) recognized by H2A antibody is H3 due to cross-reactivity of the antibody. Note, H2A-H2BssUb is recognized by anti-H2A antibody but not the anti-Ub antibody, which is likely due to epitope occlusion resulting from ubiquitin being sandwiched between H2A and H2B. (c) Composite structural model of a ubiquitylated mononucleosome (mononucleosome pdb code=1AOI, Ub pdb code=1UBQ). Ubiquitin, H2B and H2A are coloured in magenta, red and yellow, respectively. The N-terminal region of H2A, amino acids 7–10, is highlighted in cyan. Inset, close-up of the ubiquitin attachment site on H2B. (d) SDS-PAGE analysis of crosslinked reaction mixtures of mononucleosomes containing H2BssUb* and either full-length H2A, H2AΔ(1-10) or H2AΔ(1-15). (e) hDot1L methyltransferase assay on chemically defined mononucleosomes. Assays were performed on mononucleosomes with 3H-SAM and the catalytic domain of hDot1L. Quantification of methylation was performed by filter binding followed by liquid scintillation counting. Error bars indicate s.e.m. (n=3–4).
