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. Author manuscript; available in PMC: 2021 Jul 14.
Published in final edited form as: Science. 2021 Jan 22;371(6527):eabc6663. doi: 10.1126/science.abc6663

Fig. 2. Structural basis for Dot1 stimulation by H4K16 acetylation.

Fig. 2.

(A and B) Side-by-side overview and close-up of interactions of Dot1 with the H4 tail in the (A) Dot1-H4K16ac structure and (B) Dot1-unacetylated H4 structure. The structure is color-coded as in Fig. 1. The yellow dashed lines show distances of ≤3.5 Å. (C) Close-up of interactions of Dot1 with H3K79M at the catalytic site (blue arrow), the position of residues in H4 tail, and SAM. The red square indicates the region of Dot1 and H4 that is stabilized by H4K16 acetylation as shown in (A). (D) Representative endpoint methyltransferase assay for Dot1 and mutants (H347E-H355E, H335E, and I261E) in the presence of Unmod nuc or H4K16ac nuc or Ub nuc or ac/Ub nuc substrates. Reactions were performed with 0.125 and 0.250 pmol of Dot1. Each data point and error bar indicate the mean ± SD from three independent experiments. (E) Representative HMT assay measuring activity of Dot1 and its mutants on different nucleosome substrates. HMT assays were performed with an increasing amount of Dot1 mutants (H347E-H355E, H335E, and I261E) in the presence of Unmod nuc or H4K16ac nuc or Ub nuc or ac/Ub nuc substrates, and reaction products were identified by using Western blot.