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. 2020 Mar 6;11:1222. doi: 10.1038/s41467-020-15006-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — 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 hPAF15_2-11 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.