Inheritance of Histones H3 and H4 during DNA Replication In Vitro Egbert Vincent Madamba, Ellora Bellows Berthet, Nicole Jane Francis  Cell Reports  Volume 21, Issue 5, Pages 1361-1374 (October 2017) DOI: 10.1016/j.celrep.2017.10.033 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Strategy for Mapping Histones H3 and H4 from a Single Nucleosome through DNA Replication In Vitro Scheme shows steps in histone mapping starting from templates with single nucleosomes positioned over the 601 sequence in either Xenopus (middle, gray shading) or SV40 (outside, beige shading) systems. Left scheme is for detection of histones on replicated DNA, labeled with 32P during the reaction, and right scheme is for unreplicated samples that are labeled after isolation of nucleosomal DNA. See also Figures S2 and S8. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Histones Are Dispersed by DNA Replication in the SV40 DNA Replication System (A) Top blot shows biotin-tagged H3-33C and bottom blot biotin-tagged H4-63C for templates that were incubated in replication extracts but without SV40 TAg. NS indicates a sequence not present in the plasmid used as a control for background hybridization. Note that the images of the blots have been cropped and aligned, but all slots are on the same blot, and all slots from individual reactions are hybridized and exposed identically in all of the figures. Exposures for different blots in the same experiment are different, as all blots were scanned under conditions where none of the signals are saturated. (B) The same templates as in (A) after DNA replication; only the replicated templates are visible because 32P-dATP was included in the replication reaction. (C) Summary of mapping experiments. Black bars show average fraction of signal at each site for unreplicated templates; red bars are the average fraction of the signal at each site for replicated templates. See Table S1 for statistical analysis of nucleosome distribution. n = 7. Red arrows under the graph indicate the two replicons. (D–G) Analysis of histone dispersal by DNA replication using end-labeling. (D) and (E) compare blots from the same set of reactions with or without TAg; in both cases, DNA associated with biotinylated nucleosomes was end-labeled (instead of including 32P-dATP in the replication reaction) so that both replicated and unreplicated templates are visible in (E). The exposure for (D) and (E) is the same. (F) SYBR Gold-stained agarose gel (left) and blot (right) of end-labeled nucleosomal DNA isolated with and without DNA replication for reactions in (D) and (E), indicating that similar amounts are recovered in both cases. (G) Summary of 4 experiments. Bars in these and all graphs are mean ± SD. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Release and Reassembly of Biotinylated Histones Cannot Explain Nucleosome Occupancy on Replicated Templates (A) Schematics of 3 reaction paradigms. (B) SYBR Gold stained agarose gel of nucleosomal DNA recovered on streptavidin beads from different reaction schemes. (C) Summary of DNA recovery (as in B) for at least 3 experiments (using either 1X-601 or 4X-601 templates). Signals were normalized to the signal from single nucleosome templates without DNA replication (−TAg) from the same experiment. Asterisks indicate p = 0.0001, for one sample two-tailed t tests testing whether the values differ from the expected value of 1. (D) Representative blots of 4X-601 templates with preassembled templates (1) or plasmid incubated with biotinylated octamers in the replication extracts (2). (E) Summary of the distribution of biotinylated histones on 4X-601 templates in the two different reaction paradigms for at least 3 experiments. See also Figure S5. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Chromatin and Chromatin Assembly Do Not Constrain Histone Dispersal in the SV40 System (A) Schematic of experiments with hCaf-1. hCaf-1 was pre-incubated with histone octamers and added to replication extracts. (B) Hybridized blots of reactions with and without TAg in the presence of hCaf-1 and histones. (C) Summary of two experiments with hCaf1. (D and E) Summary of 3 experiments mapping the distribution of nucleosomes containing biotinylated histones using ligated templates with either naked DNA (D) or chromatinized (E) backbones ligated to single biotinylated 601 nucleosomes, with or without hCaf-1. See also Figure S7. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Distribution of Nucleosomes Containing Biotinylated H3 or H4 in Xenopus laevis DNA Replication Extracts (A) Representative maps of the distribution of nucleosomes containing biotin-H4 under different conditions. (B) Summary of nucleosome distribution prior to DNA replication (“buffer”), after incubation in HSS, and after DNA replication (HSS+NPE). See also Table S2. (C) Signals for the 601 starting location plus one position on each side are combined; p values are for two-tailed, one sample t test, comparing the value to random expectation (3/15 = 0.2). (D) Summary of distribution of nucleosomes with biotinylated H3 or H4 after incubation in HSS+NPE with or without DNA replication inhibitors. Buffer, n = 2; HSS, n = 5; replicated n = 8; aphidicolin n = 2, roscovitine, n = 5. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Release and Reassembly Cannot Explain the Distribution of Nucleosomes Containing (Parental) Biotinylated H3 or H4 on Templates in Xenopus Extracts (A) Representative blots for nucleosome mapping of reactions with 50 ng of octamers containing biotin-H3 or -H4 added to HSS with 500 ng of plasmid; ½ of this reaction was continued through DNA replication by addition of NPE. (B and C) Summary of distribution of nucleosomes with biotinylated histones from experiments mixing naked plasmid and biotinylated histones in HSS (B) or HSS+NPE (C); results from preassembled templates (Figure S9B) are included for reference. n ≥ 3. See also Figures S9C and S9D. Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Comparison of Biotinylated Histone H3 or H4 Occupancy of the 601 Sequence across Reactions and Systems (A and B) Graphs show average occupancy over the 601+/1 sequence position for the 1X-601 template (A), or the 601 sequence for the 4X-601 template (B). Red bars are for HSS+NPE, light gray and pink are plasmid + biotinylated octamers mixed in extracts. p values for (A) and (B) are from ANOVA with Tukey’s multiple comparisons test. “+ oct.” refers to addition of excess unbiotinylated octamers. (C and D) Comparison of the signal from the 601 sequence for Xenopus versus SV40 systems for 1X-601 template (601+/− one position) (C), or the 4X-601 template (D). Cell Reports 2017 21, 1361-1374DOI: (10.1016/j.celrep.2017.10.033) Copyright © 2017 The Author(s) Terms and Conditions