Nishiyama A , Mulholland CB , Bultmann S , Kori S , Endo A , Saeki Y , Qin W , Trummer C , Chiba Y , Yokoyama H , Kumamoto S , Kawakami T , Hojo H , Nagae G , Aburatani H , Tanaka K , Arita K , Leonhardt H , Nakanishi M .

             histone monoubiquitination

	Fig. 1. Dual mono-ubiquitylated PAF15 (PAF15Ub2) specifically binds to replicating chromatin.a xDNMT1 pull-downs from native chromatin extracts were analyzed by LC-MS/MS. The volcano plot summarizes the quantitative results and highlights the interacting proteins enriched upon addition of ubiquitin to UbVS-treated extracts. b Xenopus interphase egg extracts were added with sperm chromatin and incubated in the absence or presence of His6-ubiquitin (58 μM final). Chromatin-bound proteins were isolated and analyzed by immunoblotting using the indicated antibodies. For PAF15 levels in the extracts, see Supplementary Fig. 1f. c Interphase egg extracts were added with sperm chromatin and incubated in the presence of 15 μM aphidicolin (Aph) or in its absence (DMSO). Chromatin-bound proteins were isolated and analyzed by immunoblotting using the indicated antibodies. d PAF15-deleted extracts were supplemented with wild-type xPAF15-Flag3 and its variants (K18R, K27R, and K18R/K27R). After the addition of sperm chromatin, chromatin-bound proteins were isolated and analyzed by immunoblotting using the indicated antibodies. The extracts were also analyzed by immunoblotting. e PAF15-depleted extracts were supplemented with wild-type xPAF15-Flag3, its PIP-box mutant (FF/AA), or K18R/K27R mutant (KRKR). After the addition of sperm chromatin, chromatin-bound proteins were isolated and analyzed by immunoblotting using the indicated antibodies. The extracts were also analyzed by immunoblotting. Source data are provided as a Source Data file.
	Fig. 2. UHRF1 recognizes and ubiquitylates the N-terminal H3-like sequence of PAF15.a Mock-depleted or UHRF1-depleted extracts were supplemented with the indicated recombinant proteins (wt/D333A/D336A xUHRF1; see “Methods”) and chromatin was isolated. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies. For the protein levels of each protein in the extracts, see Supplementary Fig. 2a. b Comparison of the N-terminal sequence of PAF15 and histone H3 across different species. Residues mutated in the PAF15 mutants used in this study are shaded. Superimposition of plots of enthalpy changes in the interaction between hPHD and hPAF152-11 peptides by ITC measurement. c Recognition of the N-terminus of hPAF15 by hPHD. The left panel shows the crystal structure of PHD in complex with hPAF15. hPHD as a surface model with electrostatic potential (red, negative; blue, positive). The right panel shows recognition of PAF15 N-terminus (green stick model) by hPHD (pink stick model). Hydrogen bonds and water molecules are shown as black lines and balls, respectively. d PAF15-deleted extracts were supplemented with wild-type PAF15-Flag3 and its variants (R3A, T4D, and K5A). After the addition of sperm chromatin, chromatin-bound proteins were isolated after 90 min and analyzed by immunoblotting using the indicated antibodies. The level of PAF15Ub2 on chromatin was quantified for each set of conditions as explained in the “Methods” section. e, f In vitro ubiquitylation assay using the indicated hUHRF1 E3-ligases and hPAF15 substrates. Lower panels show the relative intensity of the band corresponding to dual mono-ubiquitylated PAF15. Bars represent the means of three independent experiments with SEM. Source data are provided as a Source Data file.
	Fig. 3. PAF15Ub2 forms a complex with DNMT1.a Reciprocal immunoprecipitation of PAF15 and DNMT1 from chromatin lysates. IP was performed with control (Mock), anti-xDNMT1 (DNMT1), or anti-xPAF15 (PAF15) antibody from chromatin lysates. Supernatants after immunoprecipitation (IP-sup) or immunoprecipitates (IP-ppt) were analyzed by immunoblotting using the indicated antibodies. b Sperm chromatin was replicated in interphase egg extracts containing xPAF15-Flag3 [wild-type, K18R, K27R, or K18RK27R (KRKR)]. Isolated and solubilized chromatin proteins were subjected to immunoprecipitation using anti-Flag antibodies. The resultant immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. c Superimposition of plots of enthalpy changes in the interaction between hRFTS and hPAF152-30 or its ubiquitylated analogs by ITC measurement. d Pull-down of ubiquitylated PAF15 from denatured chromatin extracts using recombinant wild-type xDNMT1-Flag3 and its ubiquitin-binding mutants (P253AL256A or I317AI362A). Source data are provided as a Source Data file.
	Fig. 4. xPAF15Ub2 promotes recruitment of xDNMT1 and maintenance of DNA methylation.a, d Sperm chromatin was added to either mock- or xPAF15-depleted extracts containing radiolabeled S-[methyl-3H]-adenosyl-L-methionine in the absence (a) or presence of 0.6 μM hRFTS (e). The efficiency of DNA methylation was measured at the time points indicated. Bar graphs depict the quantification of incorporated SAM into genomic DNA with mean and SEM from three independent experiments. Statistical significance was determined using Student’s t test. b, e Sperm chromatin was added to mock- or xPAF15-depleted interphase extracts in the absence (b) or presence (f) of hRFTS. PAF15-depleted extracts were supplemented with either buffer alone (lanes 4–6), purified wild-type xPAF15-Flag3 or K18R/K27R(KRKR) mutant xPAF15-Flag3 (320 nM final concentration, lanes 7–9 or 10–12, respectively) in the experiment described in b. At the indicated time points, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. For the PAF15 levels in extracts, see Supplementary Fig. 2a. c Sperm chromatin was replicated in mock- or PAF15-depleted interphase egg extracts. Isolated and solubilized chromatin proteins were subjected to immunoprecipitation using an anti-xDNMT1 antibody. The resultant immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. Asterisks, non-specifically detected proteins. f Schematic of experimental approach to test the differential regulation through UHRF1 during the progression of S phase. g Sperm chromatin was added to xUHRF1-depleted extracts and incubated for 0, 30, 60, 90, 120, or 150 min. Extracts were then supplemented with recombinant xUHRF1-Flag3 and further incubated for 7.5 or 15 min. Chromatin fractions were isolated and chromatin-bound proteins were analyzed by immunoblotting using the antibodies indicated. Source data are provided as a Source Data file.
	Fig. 5. Dual mono-ubiquitylation of mPAF15 is required for the mPAF15–mDNMT1 interaction in mouse ESCs.a Immunoprecipitation of endogenous DNMT1 from whole-cell lysates of wild-type J1 (WT), Dnmt1 KO (D1KO), Uhrf1 KO (U1KO), Paf15 K15R (K15R), Paf15 K24R (K24R), and Paf15 K15/24R (KRKR) mESCs using an anti-mDNMT1 nanobody. Bound fractions were subjected to immunoblotting with anti-mDNMT1 and anti-mPAF15 antibodies. The anti-mDNMT1 blot and Ponceau staining are shown as loading controls. b Immunoprecipitation of endogenous mPAF15 from WT and KRKR mESC nuclear extracts using an anti-mPAF15 antibody. Bound fractions were subjected to immunoblotting with anti-mDNMT1 and anti-mPAF15 antibodies. The anti-mDNMT1 blot and Ponceau staining are shown as loading controls. c Schematic of the fluorescent-3-hybrid (F3H) assay for the in vivo determination of protein–protein interactions. GFP-tagged bait protein is immobilized at an array of Lac operator (LacO) sequences by a GFP-binding protein (GBP) coupled to the lac repressor (LacI). When the GFP-tagged bait protein does not interact with the prey protein, only a GFP signal is visible at the LacO locus, whereas a yellow spot (combination of GFP and mCherry signal) is visible at the LacO locus in the case of a positive interaction. d, e F3H assay for a BHK cell-based analysis of ubiquitylation-mediated recruitment of mPAF15 to mDNMT1. d Cells containing a stably integrated lacO array were transfected with the GBP-LacI, a GFP-tagged bait (GFP-mDNMT1 or GFP), and an mCherry-tagged prey (mCherry-mPAF15 wild-type (WT) or mCherry-mPAF15 K15R/K24R double mutant (KRKR)). Line intensity profiles for GFP and mCherry in the respective spots are shown below the confocal images. Scale bar, 10 μm. e Quantification of the F3H assay. Background subtracted mCherry/GFP ratios within the spots were normalized to the control and plotted with n = 45 from 3 independent replicates (per replicate, n = 15). In the boxplots, horizontal black lines within boxes represent median values, boxes indicate the upper and lower quartiles, and whiskers indicate the 1.5× interquartile range. Statistical significance was determined using Student’s t test. Source data are provided as a Source Data file.
	Fig. 6. mPAF15Ub2 is required for the proper maintenance of DNA methylation in mouse ESCs.a, b DNA methylation levels (%) as measured by RRBS in wild-type (WT) and Paf15 K15R/K24R (KRKR) double mutant ESCs. a Global DNA methylation levels and b CpG methylation levels at CpG islands, promoters, gene bodies, and repeats in wt and KRKR ESCs. p Values based on ANOVA with post hoc Tukey’s test. c Density plot depicting the distribution of DNA methylation levels of individual CpG sites in wt and KRKR ESCs. d–f Replication timing of hypomethylated vs. unchanged tiles in d Paf15 KRKR ESCs, e Dnmt1 KO ESCs, and f Uhrf1 KO ESCs. For comparisons between hypomethylated and unchanged tiles, Welch’s two-sided t test was used for calculating p values. Differentially methylated tiles losing DNA methylation (hypomethylated tiles) were defined as those with p < 0.05 and a methylation loss >25%; p values were derived from a methylKit package (see “Methods”). g Model of the two pathways of dual mono-ubiquitylation facilitating maintenance of DNA methylation. Both requiring UHRF1, PAF15Ub2 and H3Ub2 preferentially contribute to the DNMT1-mediated maintenance of DNA methylation of early and late replicating regions, respectively. For the boxplots in a, b, d–f, the horizontal black lines within boxes represent median values, boxes indicate the upper and lower quartiles, and whiskers indicate the 1.5× interquartile range. Source data are provided as a Source Data file.

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