Ishiyama S , Nishiyama A , Saeki Y , Moritsugu K , Morimoto D , Yamaguchi L , Arai N , Matsumura R , Kawakami T , Mishima Y , Hojo H , Shimamura S , Ishikawa F , Tajima S , Tanaka K , Ariyoshi M , Shirakawa M , Ikeguchi M , Kidera A , Suetake I , Arita K , Nakanishi M .

	Figure 1. Dnmt1 Specifically Binds to Two-Mono-H3Ub2 (A) Mock-, Uhrf1-, or Dnmt1-depleted egg extracts were pre-treated with or without UbVS and then incubated with wild-type Ub. Sperm chromatin was added to the extracts and then isolated at the indicated times. Chromatin-bound proteins were analyzed by immunoblotting. (B) Sperm chromatin and wild-type Ub were added to untreated or UbVS-treated extracts. Isolated and solubilized chromatin proteins were subjected to a pull-down experiment using FLAG-tagged recombinant wild-type mDnmt1 coupled with anti-FLAG M2 beads. The resultant immunoprecipitates were analyzed by immunoblotting. (C) Sperm chromatin was added to Mock- or Dnmt1-depleted extracts. The isolated and solubilized chromatin proteins were subjected to immunoprecipitation using anti-H3 antibodies. The immunoprecipitates were subjected to immunoblotting using anti-Ub antibody or ubiquitin-AQUA/PRM analysis. Tryptic digests of H3Ub were quantified with ubiquitin AQUA peptides (see STAR Methods). Error bars, SD for duplicate experiments. (D) H3Ub in UbVS-treated extracts in the presence of excess amounts of wild-type or a mutant Ub in which all Ks were replaced by R (K/R). The chromatin fractions were isolated after the sperm addition and subjected to immunoblotting. (E) MS/MS spectra identifying ubiquitylations at both K18 and K23 on histone H3. The upper spectra were obtained from mDnmt1-FLAG-associated two-mono-ubiquitylated histone H3 prepared as described in Figure 1B. The lower spectra are from synthetic, isotopically labeled peptides; identified b and y fragment ions are shown. See also Figure S1. (F) FLAG-tagged recombinant wild-type xH3.2 or the indicated mutants were added to UbVS+Ub-treated extracts and analyzed as in Figure 1A.
	Figure 2. Overall Structure of hRFTS in Complex with H3-K18Ub/23Ub (A) ITC thermograms (upper) and plots of corrected heat values (lower) for binding of the hRFTS to H3-K18Ub/23Ub, H3-K14Ub/23Ub, H3-K14Ub/18Ub, ubiquitin, and H31-37W. See also Figure S2. (B) Ribbon representation of hRFTS in complex with H3-K18Ub/23Ub. N- and C-lobes, Ub18, Ub23, and the H3 tail are colored magenta, pink, cyan, green, and yellow, respectively. The enlarged figure shows disulfide-mediated ubiquitylation between G76C in Ub18 and K18C in H3, on which the |Fo| − |Fc| omit map contoured at 4 σ is superposed. See also Figure S3. (C) Schematic representation of the complex. Color schemes are the same as in Figure 2B.
	Figure 3. Binding Surface between hRFTS and K18Ub/23Ub (A) Hydrophobic interactions between hRFTS and K18Ub/23Ub. hRFTS is presented as a surface model with its electrostatic potential, in which blue and red indicate a positive and negative charge, respectively. The I44 patch of each ubiquitin, which consists of Leu8, Ile44, His68, and Val70, is shown as a stick model superposed on a transparent spherical model. The magnified figures show the interaction between hRFTS and the I44 patch of each ubiquitin. Amino acid residues of hRFTS, Ub18, and Ub23 are shown in magenta, cyan, and green stick models superposed on a transparent spherical model, respectively. See also Figure S4. (B) Hydrophilic interaction between hRFTS and K18Ub/23Ub. The left panel shows the interaction between the flexible loop and the I36 patch of Ub18 and the URL. The middle and right panels show a binding surface of Ub23 interacting with the URL and interaction between the flexible loop of Ub23 and the URL, respectively. Water molecules are colored black. (C) Purified recombinant FLAG-tagged wild-type xDnmt1, I317A/I362A, or P253A/L256A were coupled with anti-FLAG M2 beads and then incubated with MNase-digested chromatins isolated from control or UbVS-treated extracts in the presence of excess wild-type Ub. The resultant complexes and the inputs were analyzed by immunoblotting. (D) Interphase extracts depleted with either control or xDnmt1 antibodies were incubated with control buffer, purified recombinant wild-type xDnmt1, I317A/I362A, or P253A/L256A (85 nM final concentration). The chromatin fractions were isolated at the indicated times, and their bound proteins as well as inputs were analyzed by immunoblotting. See also Figure S4.
	Figure 4. Interaction Surface between the H3 Tail, hRFTS, and Ub23 (A) The N- and C-lobes and K18Ub/23Ub are shown as a surface model. The magnified figure shows the |Fo| − |Fc| omit map contoured at 3 σ superposed on the H3. The density map and H3 tail are shown as blue mesh and a yellow stick model, respectively. Trp464 shown as a pink sphere acts as a wall that kinks the H3 tail at the peptide bond between Gly12 and Gly13. See also Figure S5. (B–E) Recognition of the H3 tail by hRFTS and Ub23. Color schemes are the same as in Figure 2B. Water molecules are colored black.
	Figure 5. Enzymatic Activity of mDnmt1 (A) Dnmt1 activity was measured in the presence or absence of the indicated concentrations of H3(1-35), ubiquitin, H3 (1-35) K18Ub/23Ub, H3 (1-35) K18Ub, and H3 (1-35) K23Ub. The activity was shown as an average ± SEM (n = 3). See also Figure S6. (B) The methylation activity of Dnmt1 was measured at 25°C, 30°C, and 37°C. The logarithms of DNA methylation activities obtained (ln[DNA methylation activity], ordinate axis) against inverse temperatures (1/T, abscissa) are plotted (Arrhenius plot).
	Figure 6. hRFTS Binding to H3-K18Ub/23Ub Predicts the Opening of the Dnmt1 Catalytic Pocket (A) Structural comparison of the RFTS in hDnmt1 (left) and RFTS:H3-K18Ub/23Ub complex (right). N- and C-lobes are colored magenta and light-pink, respectively. The RFTS C-terminal linker, C-linker, and H3 are shown as blue and yellow, respectively. (B) Structural change in α4 upon the binding of H3Ub2. α4 in hDnmt1 and hRFTS:H3Ub2 complex are colored green and magenta, respectively. N- and C-terminal residues in α4 and Met502 are shown as stick models. (C) Superimposition of RFTS C-terminal linker with hRFTS:H3-K18Ub/23Ub complex. The color scheme of hRFTS:H3-K18Ub/23Ub is the same as in Figure 2B. The RFTS C-terminal linker is depicted as a blue stick model. (D) hDnmt1 structures after 100 ns simulation. The free form (left) and the complex (complex 2 as a representative structure) (right). The N-lobe and C-lobe of RFTS, and the catalytic domain are colored pink, magenta, and bright blue, respectively. The RFTS C-terminal linker, H3, and two ubiquitins (18Ub/23Ub) are colored blue, yellow, green, and cyan. Note that RFTS, the catalytic domain, and the visible part of the linker in the crystal structure are additionally shown as spheres. (E) Simulation results for the free form of Dnmt1 and in complex with H3-K18Ub/23Ub. The upper panel shows the average and the SD of Cα RMSD of the RFTS C-lobe after superimposition of the catalytic domain against the crystal structure of the free form, calculated over 100 ns trajectories. The left bar in the lower panel shows the average and the SD of the number of atom contacts, Nc, between the RFTS C-lobe and the catalytic domain, calculated over 100 ns trajectories. Nc is defined as the number of non-hydrogen atom pairs with a pairwise distance <4 à . The right bar in the lower panel shows the Nc between the RFTS N-lobe and the catalytic domain. See also Figure S6.