Volume 17, Issue 2, Pages 301-311 (January 2005) Human Asf1 Regulates the Flow of S Phase Histones during Replicational Stress  Anja Groth, Dominique Ray-Gallet, Jean-Pierre Quivy, Jiri Lukas, Jiri Bartek, Geneviève Almouzni  Molecular Cell  Volume 17, Issue 2, Pages 301-311 (January 2005) DOI: 10.1016/j.molcel.2004.12.018

Figure 1 Extracts from Cells Arrested in S Phase by HU Have an Increased Capacity to Assemble Nucleosomes onto Replicating DNA (A) HeLa cells were released 1 hr from a double thymidine block, treated with HU for 1 hr, and harvested to prepare cytosolic and nuclear extracts. For comparison, similar extracts were prepared from untreated cells at the time when the parallel cultures were received HU (S-0) and 1 hr later (S-1). The extracts were tested for nucleosome assembly onto replicating SV40 origin containing DNA in the presence of T antigen (T Ag). Lane one (input) contains untreated DNA. DNA replication was visualized by incorporation of radiolabeled nucleotides ([32]P). Total DNA was visualized by ethidium bromide staining (EtBr). The positions of plasmid DNA form I (supercoiled), form II (nicked circular), and form Ir (closed circular) are indicated. The bar diagram illustrates the level of supercoiled DNA relative to total labeled DNA. Error bars represent the deviation between two independent experiments. (B) S-1 and HU nuclear extracts obtained as in A were combined with cytosolic extract from asynchronous cells (as) and analyzed as described in (A) (lanes 2 and 3). Similarly, S-1 and HU cytosolic extracts were tested together with nuclear extracts from asynchronous cells (as) (lanes 5 and 6). (C) Western blots revealing the distribution of CAF-1 p150 and p60, HIRA, histone H3, and Asf1 (a and b) between the cytosolic and nuclear extracts analyzed in (B). The position of Asf1a and Asf1b are indicated on the right. (D) Semiquantitative Western blot analysis of histone H3 and histone H4 acetylated at lysine 12 (H4AcK12) in S-1 and HU extracts from two independent experiments with PCNA and an unspecific band (*) as loading controls, respectively (2× indicates a double input of the corresponding lysate, 1×). Similar results were obtained when comparing S-0 and HU extracts (data not shown). Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 2 hAsf1 Acts as a Histone Buffer when DNA Replication Is Perturbed (A) HeLa cell lines expressing e-H3.1 or e-H3.3 were released into S phase and treated 1 hr with HU or left untreated (S-1). The level of soluble e-H3 and endogenous H3 in whole cell extracts was estimated by Western blotting with CAF-1 p60 as a loading control (top). Complexes containing e-H3 (Flag- and HA-tagged) or Asf1a were isolated from equal amounts of whole cell extract by immunoprecipitation (IP) with anti-Flag and anti-Asf1a antibodies by using Sepharose beads and preimmune rabbit serum for control reactions, respectively. The samples were analyzed for e-H3 (HA), total H3, and Asf1 by Western blotting on the same membrane. The input corresponds to 4% of the material used for immunoprecipitation (S-1) (e-H3.1 for Flag IP and e-H3.3 for Asf1a IP). Similar results were obtained when comparing S-0 and HU extracts (data not shown). (B) Graph indicating the HU-induced increase in e-H3.1 and e-H3.1 bound chaperones. Complexes containing e-H3.1 were isolated by immunoprecipitation from HU and S-1 whole cell extracts, and the amount of e-H3.1 and coprecipitating Asf1 (a and b) and CAF-1 (both p60 and p150) were determined by semiquantitative Western blotting followed by densitometric scanning (see Supplemental Figure S2). The graph illustrates the fold increase in HU extracts relative to S-1, and error bars indicate standard error of the mean in 3 independent experiments (n = 3). (C) CAF-1 p60 or Asf1a were immunoprecipitated (IP) from identical HU or S-1 whole cell extracts from e-H3.1 cells. Preimmune rabbit serum was used in control reactions (lane 4). The samples were analyzed by Western blotting for e-H3.1 (HA), Asf1, and CAF-1 p60. The input corresponds to 3% of the material (S-1) used for immunoprecipitation. Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 3 hAsf1 Is Required for Efficient Chromatin Assembly in HU-Treated Extracts (A) Addition of hAsf1 and histones complements replication-coupled chromatin assembly in S-1 extracts. HeLa S-1 and HU cytosolic extracts were combined with nuclear extracts from asynchronous cells (as) and analyzed as in Figure 1A. Where indicated, core histones and/or recombinant Asf1 (a or b) were added to the reaction after 5 min. The amount of recombinant Asf1 added corresponds to about three times the amount provided by the cytosolic extract. (B) Asf1 (a and b) or mock depletion was carried out in HU and S-1 cytosolic extracts derived from e-H3.1 HeLa cells. The depleted extracts were combined with nuclear extract from asynchronous cells and analyzed for replication-coupled chromatin assembly (left) as in Figure 1A. Where indicated, Asf1b prebound to core histones was added to the reaction after 5 min. Western blotting was used to evaluate the extent of Asf1 (a and b) depletion (right) with Chk2 as a loading control (2× indicates a double input of the corresponding lysate, 1×). The amount of recombinant Asf1 added corresponds to about 1.5 times the amount provided by the cytosolic extract. Similar results were obtained in two independent experiments and in repair-coupled chromatin assembly assays (Supplemental Figure S5B). Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 4 HU Mobilizes hAsf1 into a Histone-Containing Complex Cytosolic extracts from HU or mock (S-1)-treated S phase cells expressing e-H3.1 were analyzed by gel filtration followed by Western blotting for e-H3.1 (HA), Asf1, p48, p60, NASP, and Chk2. Fraction numbers are indicated by bars on the top and the approximate size of eluted protein complexes is shown by arrows. The high (H) and low (L) MW hAsf1 complexes are indicated. Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 5 An Increased Level of the hAsf1-Histone-Containing Multichaperone Complex Enhances Chromatin Assembly in S Phase Extracts (A) The high (H) and low (L) MW Asf1 complexes were isolated by gel filtration of S-1 extracts and analyzed by Western blotting. (B) Repair-coupled chromatin assembly was analyzed in S-1 cytosolic extracts combined with nuclear extracts from asynchronous cells. Asf1 (a and b)-depleted S-1 extracts (lanes 2–9) were complemented with the native high (H) and low (L) MW Asf1 (a and b) complexes or recombinant Asf1 (a and b) (R), with or without histones. The level of Asf1 (a and b) in each reaction was determined by Western blot analysis of reaction aliquots (bottom). Slowly migrating His-tagged recombinant Asf1 (a and b) is marked with an asterisk. The bar diagram indicates the level of supercoiling relative to total labeled DNA. (C) GST-Asf1a binds the active multichaperone complex specifically in HU-treated cytosolic extracts. HU and S-1 cytosolic extracts were depleted for Asf1 (a and b) as in Figure 3B before they were used for pull-down experiments with GST-Asf1a or GST alone. Bound proteins were analyzed by Western blotting. The input corresponds to 5% of the starting material used for depletion. Ponceau staining was used to verify equal amounts of bead bound GST and GST-Asf1a in the reactions. Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 6 Depletion of hAsf1 Impairs S Phase Completion (A) Cell cycle profiles of U-2-OS cells treated for 48 or 72 hr with siRNA against Asf1 (a and b) or GFP. Mock indicates transfection without RNA. Two different siRNA duplexes against Asf1a were used (a1 or a2) together with a duplex against Asf1b (b1). Total cell extracts from cells harvested at 48 hr were analyzed by Western blotting for hAsf1 levels (2× indicates a double input of the corresponding lysate, 1×). Similar results were obtained in HeLa cells (data not shown). Nuclear morphology was visualized by Topo-1 DNA staining 72 hr after transfection of U-2-OS cells with siRNA against hAsf1 (a1 + b1) or GFP control and analyzed for nuclear aberrations by confocal microscopy in two independent experiments (n > 300). (B) Cell cycle profiles of siRNA or mock transfected cells released into S phase from a HU block (0 hr) in the presence of nocodazole and harvested at the time indicated above each profile. Efficient depletion of Asf1 (a and b) was verified by Western blotting (data not shown). Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)

Figure 7 Model Outlining Changes in Histone Flow Imposed by Inhibition of DNA Replication In unperturbed S phase cells, hAsf1 exists in an equilibrium between an active form and an inactive histone-free complex. Histone biosynthesis is high, yet as the active hAsf1 complex transfers histones rapidly to CAF-1 for immediate deposition, the soluble pool of histones remains low. Inhibition of DNA replication results in a concomitant blockade of both replication-coupled histone deposition and further transfer of histones to CAF-1, causing a transient imbalance between histone biosynthesis and usage. The resulting accumulation of soluble S phase histones triggers mobilization of hAsf1 into the active multichaperone complex. This histone buffer function of hAsf1 ensures that an “active” pool of histones is immediately available for chromatin restoration during repair and early phases of recovery when histone biosynthesis still remains low. Molecular Cell 2005 17, 301-311DOI: (10.1016/j.molcel.2004.12.018)