Fig. 4

Dot1p has nucleosome assembly activity and enhances chromatin remodeler activity. a Protein pull-down assay of GST-tagged Dot1 proteins against core histones. SDS-acrylamide gels were stained with Coomassie blue. The Octamer panel (top) indicates histone H2A, H2B, H3, and H4 for binding with GST (negative control), GST-Dot1, a nucleosome binding-defective mutant GST-Dot1 (101–140Δ), a histone H4 tail binding-defective mutant GST-Dot1(EDVDEΔ), and GST-Nap1 proteins. The H2A–H2B dimer (second panel) represents band size of histone H2A–H2B for each protein binding. The H3–H4 tetramer panel (third panel) indicates size of histone H3–H4 for each protein binding. The 5 μg of GST fusion proteins were incubated with 10 μg of histones in 200 μl of PDB buffer for 2 h at 4 °C. In the bottom panel, arrows indicate the following: 1, GST-Dot1p, GST-Dot1p (EDVDEΔ), and GST-Dot1p (101–140Δ) (showing a smaller size); 2, GST-Nap1p; and 3, GST. b Experimental scheme of the in vitro nucleosome assembly assay. c Assay of the ability of Dot1 to assemble nucleosomes on a fragment of pGEM-3z/601 DNA. The 147 bp DNA fragment (100 ng) was incubated without (lane 1) or with (lanes 2–9) 1:1 mass ratios of core histones. The nucleosome assembly activities of wild-type Dot1 (1.5, 2.4, and 3.2 nM; lanes 4–6) and the nucleosome binding-defective mutant, Dot1(101–140Δ) (same concentrations as Dot1; lanes 7–9) are shown, with 2 μg of GST-Nap1 used as a positive control for nucleosome assembly (lane 3). The arrow labeled with ‘nucleosome’ indicates size of assembled nucleosome. The nucleosome assembly was visualized with ethidium bromide (EtBr) staining (left) and histone H3 western blotting (center), and the data were quantified (right). The data are presented are averages of three experiments, and error bars represent the s.d. for the biological replicates. d The ability of Dot1 to stimulate chromatin remodeling does not require its methyltransferase activity. Sliding assays using the nucleosome remodeler, Chd1p, were performed with Dot1p (lanes 3–5), nucleosome binding-defective Dot1(101–140∆) (lanes 6–8), and methyltransferase activity-defective mutant Dot1 (G401R) (lanes 9–11). The graph (right) indicates the relative intensities of the central nucleosome bands versus the Dot1 protein concentration. The data presented are the averages of three experiments, and error bars represent the s.d. for the biological replicates