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Figure 4. Usp7 depletion and inhibition result in accumulation of ubiquitylated histone H3 and enhancement of Dnmt1 chromatin loading. (A) Sperm chromatin was added to either mock-, Usp7-, or Usp7/Uhrf1-double depleted extracts for 90 min. Isolated chromatin fractions as well as extracts were subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. (B) Sperm chromatin was added to either mock- or Usp7-depleted extracts for 150 min in the presence or absence of 0.1 mg/ml His6-ubiquitin. Chromatin fractions were isolated and subjected to immunoblotting using anti-histone H3 antibodies. ∆ Denotes immunodepletion of the protein indicated. (C,D) HeLa cells were infected with lentiviruses expressing tet-on shRNAs targeting Luciferase (Control), Dnmt1, Usp7, and Uhrf1, in the combinations indicated. Cells were then harvested after treatment with doxycycline (1 μg/ml) for 3 days (C) and whole cell extracts were subjected to immunoblotting using the antibodies indicated. Histones were acid-extracted from cells depleted of the indicated proteins and subjected to immunobloting using anti-histone H3 antibodies (D). xDnmt1-depleted chromatin was used as a positive control. (E) USP7 deubiquitylates histone H3 in vitro. Dnmt1/Usp7 complex deubiquitylates histone H3 either in the absence or presence of SAM. The indicated immunoprecipitates (IP) were incubated with MNase-digested chromatin containing unmodified (lane1) or ubiquitylated histone H3 in the presence or absence of S-adenosylmetionine (SAM). (F) Purified recombinant Usp7 was incubated with ubiquitylated histone H3 for 2 hrs. The reaction mixtures were analyzed by immunoblotting using the antibodies indicated. (G) Sperm chromatin was added to mock- or xUsp7-depleted egg extracts. At the time points indicated, chromatin fractions were isolated and subjected to immunoblotting using the antibodies indicated. ∆ Denotes immunodepletion of the protein indicated. Source data are available online for this figure. Image published in: Yamaguchi L et al. (2017) © The Author(s) 2017. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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