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	Figure 1. Folded histone complex of H3 and H4 is required for nuclear import in the S-phase. (a) Diagram of histones H3 and H4 illustrating the two histone domains; the N-terminal tail domain and the fold domain. (b) Nuclear import of exogenous histones requires the correct folding of histones. Trace amounts of purified FLAG-tagged histone H3 (FH3), FLAG-tagged H4 (FH4) and folded complex H3/FH4 (H3/FH4) were incorporated into Physarum macroplasmodium fragments in the early S-phase, using an untreated cell fragment as the control (Cont). The cell fragments were then harvested, and nuclei were isolated and analyzed by SDS-PAGE (Stain) and Western blotting with anti-FLAG antibody. Lane M corresponds to the molecular weight marker in (Stain) and the purified H3/FH4 complex revealed by anti-FLAG antibody in (αFLAG). (c) Trace amount of exogenous H3/FH4 complex is incorporated into nuclei. Nuclei from cell fragments untreated (−) and treated with H3/FH4 (+) were isolated, analyzed by SDS-PAGE (Stain) and Western blotting revealed with anti-H4 antibodies (αH4). The determination of the amount of exogenous histone relative to endogenous was determined by Western blotting with anti-H4 antibodies. Lane M corresponds to the molecular weight marker in (Stain) and the purified H3/FH4 complex revealed by anti-H4 antibodies in (αH4).
	Figure 2. Function of the H3/H4 amino-tail domains in nuclear import in the S-phase. (a) Nomenclature and preparation of the exogenous histone complexes. The right panel is a diagram of the different histones used to form the different complexes. The regions in blue correspond to H3 domains, in green to H4 domains, and the red stars represent the FLAG-tag epitope. The complex wt corresponds to H3/FH4, (nH4)² to nH4gH3/FH4 (duplicate of the H4 tail), (nH3)² to FH3/nH3gH4 (duplicate of the H3 tail), and Sw to nFH4gH3/nH3gH4 (swapping of the two tail domains), respectively (the histone sequences are indicated in supplementary information). The right panel shows the SDS-PAGE (Stain) and the Western blot (αFLAG) of the complexes wt, (nH4)², (nH3)² and sw, respectively. (b) Microscopic observations of the different H3/H4 complexes. Following incorporation of exogenous complexes, cell fragments were squashed, fixed, and stained with anti-FLAG antibody (FLAG) and counterstained with DAPI (DAPI). The bar corresponds to 20 μm. (c) Nuclear import of H3/H4 complexes. Physarum fragments were treated during the S-phase with exogenous histone complexes: wt (H3/FH4), (nH4)² (nFH4gH3/H4), (nH3)² (FH3/nH3gH4) and Sw (nFH4gH3/nH3gH4), respectively. Nuclei were then isolated and analyzed by SDS-PAGE (Stain) and Western blotting (αFLAG). Lane M corresponds to the molecular weight marker in (Stain) and the purified H3/FH4 complex revealed by anti-FLAG antibody in (αFLAG).
	Figure 3. Exogenous histone complexes are stably incorporated into Physarum cells. (a) Hydroxy-urea treatment inhibits nuclear import. Cell fragments in early S-phase were untreted (−) and treated (+) with exogenous H3/FH4, and untreated (−HU) and treated with hydroxyl-urea (+HU), concomitantly. Nuclei were prepared and analyzed by SDS-PAGE (Stain) and Western blotting (αFLAG). Lane M corresponds to the molecular weight marker in (Stain) and the purified H3/FH4 complex revealed by anti-FLAG antibody in (αFLAG). (b) Determination of the stability of exogenous histone complexes in Physarum. Cell fragments were treated with HU and exogenous complexes were incorporated for 15 min, 30 min, 45 min and 60 min, respectively. Shown is the quantitative analysis of Flag signal relative to the amount of total soluble proteins determined by dot blotting. The quantification at time point 60 min was arbitrary assigned to 1.0 for each complex. Note that signals of untreated cell fragments with exogenous histones were ~10%. (c) Exogenous histone complexes are transported in nuclei with similar rates. The different histone complexes were spread onto Physarum surfaces and harvested after 20 min, 40 min and 60 min, respectively. Nuclei were then prepared and analyzed by Western blotting. Shown is the amount of exogenous histone complexes in the nuclear fractions at specific incorporation duration. The value 1 for each complex corresponded to the incorporation after 60 min.
	Figure 4. Chromatin assembly in the S-phase occurs regardless of histone tail positioning. (a) Microscopic distribution mapping of exogenous H3/H4 complexes. Following exogenous H3/H4 incorporation, nuclei were prepared, chromatin was combed, immuno-stained with anti-FLAG (FLAG) and counterstained with DAPI (DAPI). The chromatin fibers were imaged by fluorescent microscopy. (b) Exogenous H3/H4 complexes assembled in nucleosomes. Following incorporation, nucleosomes were isolated from nuclei by MNase digestion and sucrose gradient. Nucleosomal proteins were analyzed by SDS-PAGE (Stain) and Western blotting revealed with anti-FLAG antibody (αFLAG). Lane M corresponds to the molecular weight marker in (Stain) and the purified H3/FH4 complex revealed by anti-FLAG antibody in (αFLAG). (c) Histone stability in chromatin. Protamine competition assays were performed on nuclei and analyzed by SDS-PAGE (Endogenous histones) and Western blotting (Exogenous histones), respectively.
	Figure 5. Replication-coupled chromatin assembly requires properly positioned amino-tail domains for transferring H3/H4 from the nuclear import chaperone to the chromatin assembly complex. (a) Chromatin assembly in the S-phase occurs by replication-coupled and replication-independent chromatin assembly, which can be discriminated by EdU pulse as depicted in the diagram. Following exogenous H3/H4 complex incorporation, click reactions were performed to couple biotin to EdU and ChIP was carried out with avidin. Crosslinks of immuno-precipitated chromatin were reversed and proteins analyzed by Western blotting. Input fractions were examined with anti-H3 antibody (H3) and anti-FLAG antibody (FLAG), respectively. Immuno-precipitated fractions (ChIP EdU) were examined with anti-FLAG antibody (FLAG). (b) Replicon-specific analyses of the H3/H4 tail requirement in replication-coupled chromatin assembly and in replication-independent chromatin assembly. The top diagram represents the experimental strategy for examining replication-coupled and replication-independent chromatin assembly at specific loci. (Left gels) Physarum was pulsed for 1 h with EdU and replicated DNA was isolated by IP with avidin. Immuno-precipitated DNA was analyzed by PCR with primers specific to Lav1.1 (early replicon) and Lav2.1 (late replicon), respectively. (Right graph) Following exogenous complex incorporation, ChIP analyses were carried out using anti-FLAG antibody and qPCR with primers specific to the early replicon (Lav1.1) and the late replicon (Lav2.1), respectively. The graph represents the ΔΔCt = ΔCt (ChIP Late replicon normalized) −ΔCt (ChIP Early replicon normalized) for the different exogenous complexes. Value > 0 corresponds to preferential replication-coupled chromatin assembly (RC) and values < 0 corresponds to preferential replication-independent chromatin assembly (RI), respectively.

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