1. Volume 37, Issue 5, Pages 736-743 (March 2010)
Replication Stress Interferes with Histone Recycling and Predeposition Marking of New Histones 
Zuzana Jasencakova, Annette N.D. Scharf, Katrine Ask, Armelle Corpet, Axel Imhof, Geneviève Almouzni, Anja Groth 
Molecular Cell 
Volume 37, Issue 5, Pages (March 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 37, 736-743DOI: (10.1016/j.molcel.2010.01.033)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 Characterization of Asf1 S Phase Complexes
(A) Strategy for cell synchronization (right) and purification of e-Asf1 complexes (left). Cells were harvested in mid-S phase (S) or after 1.5 hr HU treatment (S + HU) for FACS analysis of DNA content and complex purification.
(B) Coomassie staining of e-Asf1b complexes. Recombinant Asf1a (rAsf1a) was used to estimate quantity. Mass spectrometry identified the indicated proteins. MCM2, -4, -6, and -7 were described (Groth et al., 2007a). For e-Asf1a complexes, see Figure S1.
(C) Analysis of e-Asf1a and e-Asf1aV94R complexes.
(D) Overview of Asf1-H3-H4 interactors and their cellular distribution.
(E) Gel filtration analysis of nuclear extracts from HeLa S3 cells in S phase. Two separate Asf1 complexes are indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 2 Profiling of Marks on Histones H3-H4 in Asf1b Complexes
Histones H3 and H4 were purified from cytosolic (cyt) and nuclear (nuc) Asf1b complexes as shown in Figure 1B. (A–E) Analysis by quantitative mass spectrometry. All graphs show averages of four biological replicates with error bars indicating SEM. For further details, see Supplemental Information, Figures S5–S12 and Tables S1 and S2.
(A) Quantification of acetylation on the H4 peptide aa 4–17. The major diacetylated species, K5K12diAc, was identified by MS/MS (Table S1 and Figure S7).
(B) Quantification of histones H3.1 and H3.3.
(C) Quantification of modifications on the H3 peptide aa 54–63. MS/MS identified K56ac (Figure S12).
(D) Quantification of modifications on the H3 peptide aa 18–26. MS/MS identified the acetylated species as mainly H3K18ac (Table S2 and Figure S10).
(E) Quantification of modifications on the H3 peptide aa 9–17. MS/MS identified the acetylated species as mainly H3K14ac and the methylation as H3K9me1 (Table S2 and Figure S9).
(F) Western blot analysis of histone PTMs. Asf1 complexes were analyzed in parallel with serial dilution of chromatin pellet from the same number of S phase cells. The antibodies did not cross-react with unmodified histones (Figure S2).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 3
Asf1 Is Retained with MCM2-7 on Chromatin upon Replication Arrest
(A) Analysis of Asf1 in cytosolic and nuclear extracts from cells treated as in Figure 1A. 2x indicates a double input of the corresponding lysate, 1x.
(B) Analysis of Asf1 in detergent-soluble and insoluble (chromatin-bound) fractions. Cells were treated as in (A) and fractionated as shown. Histone H4 was used as a loading control for chromatin.
(C) Analysis of chromatin-bound Asf1b complexes. Following extraction of soluble proteins, chromatin-bound proteins were released by DNase I and e-Asf1b complexes isolated under high-stringency conditions.
(D) Analysis of Asf1b and MCM2 localization by confocal microscopy. HeLa S3 cells were pre-extracted with 0.5% Triton on ice prior to fixation and immunofluorescence staining. (Top) Representative field of cells in mid-S phase treated with or without HU for 2 hr. Scale bar, 20 μm. (Bottom) Representative S phase cell treated with HU for 2 hr. Scale bar, 5 μm. Specificity of the Asf1b antibody was confirmed in RNAi experiments (Figure S3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 4
Replication Stress Challenges Histone Recycling and Predeposition Marking
(A) (Top) Time course analysis of ssDNA by immunoflourescence. Scale bar, 50 μm. (Bottom) Colocalization of ssDNA and CAF-1 p60 using confocal microscopy. A representative mid-S phase cell is shown. Scale bar, 5 μm. HeLa S3 cells were prelabeled with BrdU, synchronized in S phase, and treated with HU as in Figure 1A. To reveal ssDNA, we exploited that the BrdU epitope is accessible in ssDNA and occluded in dsDNA. Some ssDNA foci also contained Asf1b (Figure S4).
(B) Quantification of ssDNA by semiquantitative dot blot. DNA from cells prelabeled with BrdU and treated with HU was analyzed under native conditions. BrdU in ssDNA was detected by western blotting. Error bars represent standard deviation of two independent experiments performed in triplicates.
(C) Time course analysis of PTMs in Asf1b complexes after HU treatment. We purified nuclear e-Asf1b complexes from cells in S phase (0) and after HU treatment as indicated. Chromatin from S phase cells was included for comparison.
(D) Asf1 recycles histones during recovery. We purified e-Asf1b complexes from S phase cells treated as indicated. For recovery (R), HU was washed out and cells were allowed to progress in S phase for 1.5 hr.
(E) Analysis of H3K9me1 in Asf1b complexes (left) and nuclear extracts (right) during recovery from HU. Cells were treated as in (D).
(F) Comparison of H3K9me1 in Asf1b complexes from early and late S phase cells challenged with replication stress. (Left) FACS analysis of synchronized cells. (Right) Western blot analysis. 2x indicates a double input of the corresponding complex, 1x.
(G) Hypothesis. Replication stress is known to promote genetic instability and cancer (Halazonetis et al., 2008). We show here that acute replication arrest interferes with normal histone dynamics and triggers accumulation of H3K9me1 and evicted histones in Asf1 complexes. We hypothesize that unscheduled incorporation of such premarked histones could present a hazard to chromatin restoration and potentially introduce epigenetic changes in cell progeny.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions