1. Volume 16, Issue 6, Pages 979-990 (December 2004)
Binding of Chromatin-Modifying Activities to Phosphorylated Histone H2A at DNA Damage Sites 
Jessica A. Downs, Stéphane Allard, Olivier Jobin-Robitaille, Ali Javaheri, Andréanne Auger, Nathalie Bouchard, Stephen J. Kron, Stephen P. Jackson, Jacques Côté 
Molecular Cell 
Volume 16, Issue 6, Pages (December 2004)
DOI: /j.molcel

2. Figure 1
Histone H2A Is Phosphorylated at Serine 129 Near a DNA Double-Strand Break In Vivo
(A) Chromatin immunoprecipitation (ChIP) assay of H2A phosphoserine-129 upon induction of a single double-strand break in vivo. JKM115 cells transformed with galactose-inducible HO nuclease encoding plasmid (pGAL-HO) or empty vector (pDAD1) were incubated in glucose or galactose media for 45 min and processed for ChIPs with purified anti-H2A P-Ser129. Precipitated DNA was analyzed by PCR with primers corresponding to chromosomal regions next to the HO cutting site, 1.5 kb and 10 kb downstream or an unrelated locus (GEA2). H2A P-Ser129 signal is specifically increased around the HO site after cleavage by the nuclease.
(B) A similar experiment with JKM179 cells (integrated GAL-HO gene) and quantification of ChIP results by real-time PCR. Occupancy is measured as the ratio of IP at the HO sites compared to a control locus (see Experimental Procedures). Data is presented as change of that ratio after 45 and 90 min where time 0 is set to 1. In this experiment, >90% of the HO site is cut after 45 min in galactose.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
The NuA4 Complex Interacts Directly with Histone H2A P-Ser129 through Its Arp4 Subunit
(A) H2A peptide pull-down assay with protein extract from yeast cells. Whole cell extract (input), matrix bound (beads), H2A bound, or H2A P-Ser129 bound fractions were analyzed by Western blotting with anti-Esa1 and anti-Arp4 antibodies.
(B) Analysis of HAT activity in samples prepared as in (A) with free histones as a substrate.
(C) Assays were performed as in (A) with purified NuA4 complex.
(D) H2A peptide pull-down assays with purified recombinant NuA4 subunits were performed as in (A). Western blots were probed for the indicated proteins.
(E) H2A peptide pull-down assays with NuA4 complexes purified from wild-type and arp4-12 mutant strains were analyzed by Western blot with anti-Esa1.
(F) HAT activity analysis of samples prepared as in (E).
(G) Arp4 preferentially binds P-Ser129-containing peptides. Peptide pull-down assays were done as in (D) with recombinant Arp4 and different phosphopeptides corresponding to H2A C terminus (P-Ser122, P-Ser129) or H4 N terminus (P-Ser1).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
The NuA4 Complex Is Important for DNA Repair and Accumulates Near a DNA Double-Strand Break In Vivo in an Arp4-Dependent Manner
(A) Cells harboring mutations in NuA4 components or its substrates are highly sensitive to DNA damage. 10-fold dilutions of mid-log cultures of esa1-Δ414, hta1-ΔN(H2A), hhf2-ΔN(H4), arp4-3, arp4-12, arp4-26, arp4-S23A mutant, and isogenic wild-type yeast strains grown in the presence or absence of 0.03% MMS.
(B) Esa1 is recruited to a double-strand break in vivo concurrently with the detection of histone H2A phosphorylated Ser129. Chromatin immunoprecipitations (ChIPs) were performed with anti-Esa1, anti-H2A P-Ser129, or preimmune serum. pGAL-HO- or pDAD1-transformed strains described in Figure 1A were grown in the presence or absence of galactose for 45 min. Upon induction of the HO nuclease, precipitated material by anti-Esa1 and anti-H2A P-Ser129, but not preimmune serum, is specifically enriched in the chromatin proximal to the double-strand break. Signal from anti-H2A P-Ser129, but not from anti-Esa1, is detected 10 kb away from the DSB.
(C) ChIP assays as above with strains containing a C-terminal TAP tag inserted at the EPL1 locus. In this case, IgG-Sepharose and anti-H2A P-Ser129 were used to precipitate chromatin. As for Esa1, the NuA4 subunit Epl1 is enriched near the DSB after HO nuclease induction, whereas no effect is seen at an unrelated chromosomal locus (GEA2).
(D) Real-time PCR/ChIP experiment as in Figure 1B with antibodies against Arp4, a protein found in NuA4 and other chromatin modifying complexes (see text). Arp4 occupancy is measured at 2 and 4 hr of HO nuclease induction (>90% cutting of HO).
(E) Similar experiment using antibodies against Eaf1, a protein found exclusively in the NuA4 complex (A. Auger et al., submitted). NuA4 occupancy is measured at 45 and 90 min of HO nuclease induction (>90% of HO site cleaved).
(F and G) Arp4 is required for NuA4 binding near a DSB in vivo. Real-time PCR/ChIP analysis with wild-type and arp4-12 mutant cells grown at room temperature with anti-Eaf1 and anti-H2A P-Ser129. HO endonuclease was induced for 45 min in galactose media (80% and 50% cutting of HO in wt and mutant cells, respectively).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Arp4 Is an H2A Phosphoserine-129 Recognition Interface in Multiple Chromatin Modifying/Remodeling Complexes
(A) Tandem affinity purification of Arp4 shows association of numerous polypeptides. Purified material from untagged and Arp4-TAP strains were loaded on gel and visualized by silver staining. Bands corresponding to the NuA4 subunits are indicated on the right (italicized). Specific subunits of the Ino80 and Swr1 complexes are also labeled and underlined, including the Rvb1/2 helicases. Arp4 and Act1 are present in all three complexes. Rvb1/2 are present in both Ino80 and Swr1 complexes, whereas Eaf2(Swc4) and Yaf9 are in NuA4 and Swr1.
(B) Ino80 specifically interacts with H2A P-Ser129 in cell extracts. H2A peptide pull-down assay as in Figure 2 with whole cell extract prepared from a Myc-tagged INO80 strain.
(C) Ino80 is recruited to a DNA double-strand break in vivo. ChIP assays were performed as in Figure 1A with a TAP-tagged INO80 strain. The HO nuclease was induced for 0, 45, or 90 min in galactose before cross linking. PCR analysis of the chromatin precipitated by the IgG beads shows that Ino80 amount increases with time near the HO-cutting site. Ino80 is detected 1.5 kb away from the DSB but is not enriched at other genomic loci (GEA2, POL1).
(D) Rvb1, a subunit of Ino80 and Swr1 complexes, accumulates near a DSB in vivo. Realtime PCR/ChIP analysis as in Figure 1B with anti-Rvb1, anti-H2A P-Ser129, anti-Eaf1, and anti-H3 triMe-Lys4. HO nuclease was induced for 45 min in galactose media before crosslink.
(E) Accumulation of Ruvb1 near a DSB requires Arp4. Real-time PCR/ChIP experiments with wt and arp4-12 mutant cells grown at room temperature. Upon induction of the nuclease for 45 min, increased binding of Rvb1 and Eaf1 near the HO DSB is only detected in the wild-type cells (HO cleavage is >90% in wt and 82% in mutant cells).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
The Swr1/Htz1 Complex Binds H2A Phosphoserine-129 In Vitro and Functionally Interacts with It In Vivo
(A) H2A peptide pull-down assay with purified SWR1, NuA4, and SAGA multisubunit complexes were performed as in Figure 2. Western analysis of the bound fractions were done with anti-Rvb1 for SWR1 and anti-Tra1 for NuA4 and SAGA (subunit shared between the two complexes). As for NuA4, the SWR1 complex interacts specifically with P-Ser129 peptides in vitro, whereas the Arp4-less SAGA complex does not bind.
(B) swr1 and htz1 mutant cells are sensitive to DSB-inducing agent camptothecin and show genetic interaction with histone H2A Ser fold dilutions of mid-log cultures were analyzed as in Figure 3A on YPD plates with or without 10 μg/ml of camptothecin.
(C) Sensitivity of htz1 mutant cells to camptothecin is suppressed by overexpression of Arp4 in a H2A Ser129-dependent manner. Growth analysis as in (B) by using htz1 and htz1/H2A-S129A mutant cells transformed with a high copy plasmid overexpressing Arp4 or an empty vector. 30 μg/ml of camptothecin was used in this experiment.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Chromatin Modifiers/Remodelers Are Recruited in an Ordered Fashion Near a DSB In Vivo
A time course experiment was performed by incubating the cells in galactose-containing media for 0, 15, 30, 45, 60, 120, or 240 min before crosslinking/ChIP and analysis by real-time PCR. (A) Phosphorylation of H2A Ser129 occurs rapidly and achieves high levels within 30 min of HO induction. (B) NuA4 (Eaf1) is also recruited rapidly and achieves maximum levels within 60 min of HO induction. (C) Rvb1-containing chromatin remodeling complexes are recruited at a later stage after DSB formation. Significant increase of Rvb1 signals is only detected after 120 min of HO induction. HO cleavage at the different time points of induction is 48%, 79%, 79%, 83%, 90%, and 91% (data not shown).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Transient Increase of Histone H4 Acetylation in the Vicinity of a DSB Is Required for Efficient Recruitment of Rvb1 Helicase-Containing Complexes
(A and B) The chromatin prepared in Figure 6 was used in ChIP with antibodies against H2A P-Ser129, total histone H2A (acidic patch), and acetylated lysine 8 of histone H4. Signals were corrected for possible nucleosome loss within 1.5 kb of the HO DSB break by normalizing with total histone H2A. (A) Kinetics of H2A phosphorylation close to the DSB shows a rapid increase to maximum levels within 30 min followed by an apparent decrease at later time points. (B) Histone H4 is rapidly acetylated within 15 min of HO break induction and seems to become deacetylated close to the break after 120 min.
(C and D) A time course experiment was performed by incubating wild-type and esa1-L254P mutant cells at 28°C in galactose-containing media for 0, 0.5, 1, 2, or 4 hr before crosslinking/ChIP and analysis by real-time PCR. In these conditions, the mutant cells are viable but show a defect in Esa1-dependent histone H4 acetylation (Clarke et al., 1999) (data not shown). In wild-type cells, Rvb1-containing chromatin remodeling complexes are recruited near the DSB in a kinetic similar to what was seen in Figure 6C, both at the break (C) and up to 1.5 kb away from it (D). This recruitment is clearly defective in esa1 mutant cells. HO cleavage at the different time points of induction is 89%, 94%, 94%, and 94% in wild-type cells whereas 55%, 80%, 84%, and 84% in mutant cells (data not shown).
Molecular Cell  , DOI: ( /j.molcel )