1. Volume 144, Issue 2, Pages 200-213 (January 2011)
Global Regulation of H2A.Z Localization by the INO80 Chromatin-Remodeling Enzyme Is Essential for Genome Integrity 
Manolis Papamichos-Chronakis, Shinya Watanabe, Oliver J. Rando, Craig L. Peterson 
Cell 
Volume 144, Issue 2, Pages (January 2011)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Global Alterations in the H2A.Z Nucleosomal Incorporation upon Disruption of the INO80 Complex
(A–C) Increased misincorporation of H2A.Z and compression of the H2A.Z-enrichment range in the absence of INO80 as measured by mononucleosomal ChIP-chip analysis. (A) Distribution of the log2 median ratios of H2A.Z enrichment in WT and ino80 nucleosome populations. Value 0 on the x axis reflects the average H2A.Z enrichment in each strain. (B) Scatter plot analysis comparing H2A.Z enrichment in WT versus ino80 mutant. Blue dots represent H2A.Z nucleosomes. The trendline equation indicates the global change of the distribution of H2A.Z in the ino80 strain compared to WT. (C) Heatmap from a mononucleosomal ChIP-chip experiment for H2A.Z conducted in WT and ino80 cells. Each box represents 140 nucleotides. Squares in the same column represent the same nucleosomes. Green signal indicates H2A.Z enrichment below average and red signal indicates positive enrichment. Black indicates average enrichment. Scheme represents the respective scanned region of chromosome III.
(D) Equal levels of chromatin-bound H2A.Z in WT and ino80 strains. Chromatin fractions were isolated from WT and ino80 cells and immunoblot analysis was performed for the indicated proteins. Similar results were found when nucleosomal histones were released from the chromatin pellet by MNase digestion prior to the SDS-PAGE analysis (data not shown).
(E and F) Pronounced mislocalization of H2A.Z at RNA PolII genes. (E) Scatter plot analysis comparing H2A.Z enrichment at promoter nucleosomes in the indicated strains. Analysis conducted as in (B). (F) Distribution analysis of H2A.Z enrichment at the first nucleosome downstream of TSS (+1 nucleosomes) and in the coding sequence (mid- and 3′ CDS) in WT and ino80 strains. Analysis conducted as in (A).
See also Figure S1.
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4. Figure 2 H2A.Z Is Not Lost from Nucleosomes in the Absence of INO80
(A and B) Scatter plot analyses comparing H2A.Z enrichment in G2/M- versus G1-arrested WT (A) and ino80 mutant (B) cells.
(C) Indicative heatmaps of H2A.Z nucleosomal occupancy at the KAR4 locus under repressed (G2/M) and activated (G1) conditions in the indicated strains.
(D and E) Nucleosome positioning and nucleosome loss at the KAR4 promoter. (D) Representative graph demonstrating nucleosome positioning in WT and ino80 cells at the indicated conditions as measured by amplification of genomic mononucleosomal DNA by qPCR. Values reflect the ratio of the amplified tested DNA over the total DNA purified from mononucleosomes. Scheme represents the promoter and coding region of KAR4. Numbers of the nucleosomes are relative to the TSS. (E) Nucleosome loss was measured from (D) as the fold decrease of the DNA amplified in repressed overinduced conditions after correction of the ratios of amplification achieved with total MNased DNA.
(F and G) H2A.Z is not evicted from the KAR4 promoter during transcriptional induction in the ino80 strain. (F) Mononucleosomal-ChIP assay for H2A.Z was conducted in the WT and ino80 strains in the indicated conditions. Values reflect the average absolute amplification of the tested DNA from three independent experiments. (G) Mononucleosomal-ChIP assay for H2A.Z was conducted in the WT and ino80 strains in the indicated conditions. Values reflect the average enrichment of H2A.Z normalized to the respective input DNA from three independent experiments. Each bar represents the average enrichment of the tested DNA from six primer pairs inside the corresponding nucleosomal region.
See also Figure S2.
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5. Figure 3 INO80 Has ATP-Dependent Histone-Exchange Activity
(A) Scheme of in vitro dimer-exchange assay with recombinant yeast mononucleosomes. Mononucleosomes were incubated with remodeling enzymes and free dimers. Incorporation of dimers into the nucleosomes was analyzed by native PAGE and western blotting.
(B) SWR-C incorporates H2A.Z/H2B dimers into H2A-containing nucleosomes. H2A-containing mononucleosomes (100 nM) were incubated with the indicated remodeling enzymes (5 nM) and free, HA-tagged H2A.Z/H2B dimers (50 nM) in the presence or absence of ATP.
(C) INO80 incorporates H2A/H2B dimers into H2A.Z-containing nucleosomes. H2A.Z-containing mononucleosomes (100 nM) were incubated with the indicated remodeling enzymes (5 nM) and free FLAG-tagged H2A/H2B dimers (50 nM) in the presence or absence of ATP.
(D) Concentration dependence of INO80 on H2A/H2B dimer incorporation activity. Increasing amounts (3, 6, and 12 nM) of INO80 was used as in (C).
(E) INO80 catalyzes a histone-exchange event. HA-tagged, H2A.Z-containing mononucleosomes (100 nM) were incubated with INO80 (5 nM) and free FLAG-tagged H2A/H2B dimers (50 nM) in the presence or absence of ATP.
(F) INO80 incorporates H2A/H2B dimers into H2A.Z-containing nucleosomes. Streptavidin bead-immobilized H2A.Z-containing, biotinylated mononucleosomes (100 nM) were incubated with INO80 (5 nM) and free FLAG-tagged H2A /H2B dimers (50 nM) in the presence or absence of ATP. After washing, incorporation of dimers into the nucleosomes was analyzed by SDS-PAGE and western blotting. H2B values reported in this experiment reflect the standard deviation from three independent experiments.
(G) INO80 does not incorporate H2A/H2B dimers into H2A-containing nucleosomes. H2A-containing biotinylated mononucleosomes (100 nM) were incubated with remodeling enzymes (5 nM) and free FLAG-tagged H2A/H2B dimers (50 nM) in the presence or absence of ATP.
See also Figure S3.
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6. Figure 4
Reduced H2A.Z Expression Suppresses the Replication Defects of the ino80 Mutant Strain
(A) Left: Schematic representation of the HTZ1 locus carrying the truncated HTZ1 promoter. Right: Reduced H2A.Zp levels from the crippled promoter. Acid-extracted proteins from logarithmically grown cells were separated by SDS-PAGE and immunoblot analysis was performed for H2A.Z. Equal gel loading was confirmed by western blotting against histone H3.
(B) Reduced incorporation of HTZ1cp in chromatin. Chromatin fractionation assay in WT, HTZ1cp and HTZ1cp, and ino80 strains and total and chromatin fraction of proteins were separated by SDS-PAGE and analyzed by immunoblotting for H2A.Z. The asterisk (∗) indicates an apparent cytoplasmic protein that cross-reacts with the anti-H2A.Z antibody and serves as a fractionation control.
(C) Expression of HTZ1cp promotes replication during replication stress conditions in the absence of INO80. Cells from the indicated strains were synchronized in G1 phase with alpha factor (αF) and subsequently released into nocodazole-containing YPD media with 40 mM HU. Cell samples were collected at the indicated times and analyzed for DNA content by flow cytometry analysis.
See also Figure S4.
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7. Figure 5 Functional Interactions between INO80 and H2A.Z Acetylation
(A) Decrease of H2A.Z-K14 acetylation in the ino80 mutant is dependent on Hda1. Acid-extracted proteins from WT, ino80, hda1, and ino80 hda1 double-mutant cells were separated by SDS-PAGE and assayed for total and K14 acetylated H2A.Z by western blotting. Equal gel loading was confirmed by immunoblot analysis against Eaf3.
(B) H2A.Z acetylation is essential for the viability of the ino80 mutant cells in DNA damage and replication stress conditions. WT, HTZ1-K(3,8,10,14)R (HTZ1 4KR), ino80, and ino80 mutant cells expressing the HTZ1-K(3,8,10,14)R derivative (HTZ1 4KR,ino80) were plated in 10-fold serial dilutions on YPD plates containing the indicated concentration of HU or zeocin to induce DNA replication stress or DNA DSBs, respectively. Pictures of the plates were taken after 2–5 days of incubation at 30°C.
(C) Deletion of HDA1 suppresses the replication defects of the arp8 strain during replication stress conditions. Log-phase cells from the indicated strains were plated in 10-fold serial dilutions on YPD plates containing 20 mM HU. Pictures of the plates were taken after 2–4 days of incubation at 30°C.
See also Figure S5.
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8. Figure 6
The Genomic Instability Phenotypes Caused by Disruption of the INO80 Complex Are Rescued by the H2A.Z-K(3,8,10,14)Q Acetylation Mimic Mutant
(A) WT, arp8, and arp8 mutant cells expressing the HTZ1-K(3,8,10,14)Q allele (HTZ1 4KQ,arp8) were synchronized in G1 phase with alpha factor (αF) and subsequently released into nocodazole-containing YPD media with 40 mM HU. Cell samples were collected at the indicated times and analyzed for DNA content by flow cytometry analysis.
(B) WT and arp8, ino80, or arp5 single mutant cells ectopically expressing from the URA3 locus either the WT (HTZ1) or the HTZ1-K(3,8,10,14)Q (HTZ1 4KQ,arp8) allele were plated in 10-fold serial dilutions on YPD plates containing the indicated concentration of HU, methylmethanesulfonate (MMS) or zeocin to induce DNA replication stress, DNA damage during replication, or DNA DSBs, respectively. Pictures of the plates were taken after 2–5 days incubation at 30°C.
(C) RT-qPCR analysis of INO1 mRNA isolated from the indicated strains grown either in the presence of inositol [yeast extract, peptone, dextrose (YPD)] or for 2 hours in synthetic media lacking inositol (−inositol). The values of INO1 mRNA were normalized to the respective ACT1 transcripts and normalized INO1 transcript in WT cells grown in the absence of inositol was arbitrarily set as 100. Error bars represent variation between two separate experiments.
(D) Cells from the indicated strains were plated in 10-fold serial dilutions on YPD plates or plates containing synthetic media lacking inositol. Pictures of the plates were taken after 2–4 days incubation at 30°C.
(E) Resistance of the HTZ1-K(3,8,10,14)Q arp8 cells to genomic instability-inducing agents is due to the HTZ1-K(3,8,10,14)Q locus. pHTZ1/arp8, htz1Δ and pHTZ1/arp8, htz1-4KQΔ strains were plated in 10-fold serial dilutions on YPD plates containing the indicated concentration of HU and zeocin. Pictures were taken after 2–4 days days incubation at 30°C.
(F) Nucleosome and H2A.Z loss during transcriptional induction are similar in the HTZ1 arp8 (arp8) and htz1-K(3,8,10,14)Q arp8 (htz1-4KQ arp8) strains. Representative graphs demonstrating nucleosome and H2A.Z loss at the KAR4 promoter in the indicated strains. The fold decrease rates were measured as in Figure 2E. (G) Similar high enrichment of H2A.Z and H2A.Z-K(3,8,10,14)Q at the KAR4 locus in the absence of arp8. Mononucleosomal-ChIP for H2A.Z was conducted in chromatin from the indicated strains arrested in G1 by alpha factor. Analysis was conducted as in Figure 2G.
See also Figure S6.
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9. Figure 7
Proposed Model for the Role of the INO80 Chromatin-Remodeling Complex in Establishing the Proper Chromatin Localization of H2A.Z
In this model, INO80 performs a boundary function by removing H2A.Z that is deposited distal to the targeted SWR-C enzyme. See text for details.
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10. Figure S1
Global Alterations in the H2A.Z Nucleosomal Incorporation upon Disruption of the INO80 Complex, Related to Figure 1
Indicative heatmaps from a mononucleosomal ChIP on chip experiment for histone H2A.Z conducted in WT and ino80 cells. Squares in the same column represent same nucleosomes. Green signal indicates H2A.Z enrichment below average and red signal indicates positive H2A.Z enrichment. Black indicates average H2A.Z enrichment. Scheme represents the respective scanned regions of chromosome III.
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11. Figure S2
H2A.Z Is Not Lost from Nucleosomes in the Absence of INO80, Related to Figure 2
(A) Expression of KAR4 is normal in the ino80 mutant strain. RT-qPCR analysis of KAR4 mRNA isolated from WT and ino80 cells arrested in G1 phase by alpha factor. The values of KAR4 mRNA were normalized to the respective ACT1 transcripts and normalized KAR4 transcript in WT cells was arbitrarily set as 100. Error bars represent s.d. between two separate experiments.
(B) H2A.Z is not evicted from the KAR4 promoter during transcriptional induction in the ino80 strain. Mononucleosomal-ChIP assay for H2A.Z was conducted in the WT and ino80 strains in the indicated conditions. Values reflect the fold enrichment of H2A.Z in each condition normalized to the levels of H2A.Z enrichment in WT in G1 for each tested DNA after correction to the respective input DNA. Each bar represents the average enrichment of the tested DNA from six primer pairs inside the corresponding nucleosomal region. Error bars represent s.d. from three independent experiments.
(C) Alterations in the H2A.Z nucleosomal incorporation upon disruption of the INO80 complex in non cell-cycle regulated genes. Indicative heatmaps from a mononucleosomal ChIP on chip experiment for histone H2A.Z conducted in WT and ino80 cells in G1 (alpha factor arrest) and in G2/M (nocodazole arrest) conditions. Analysis was conducted as in Figure S1.
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12. Figure S3
In Vitro Activities of INO80 and SWR-C-Remodeling Enzymes, Related to Figure 3
(A) INO80 does not evict histone octamers in vitro. Mononucleosomes were reconstituted with recombinant yeast histone octamers and [α-32P]dCTP-labeled 200 bp DNA fragment containing the 601 sequence. Mononucleosomes were incubated with remodeling enzymes and supercoiled plasmid DNA as a histone acceptor. Following incubation, reactions were electrophoresed on native PAGE. In this assay, SWI/SNF showed robust histone eviction activity for both H2A-containing and H2A.Z-containing mononucleosomes as indicated by the loss of the mononucleosome species and increased free DNA. Note lack of activity for INO80.
(B) INO80 mobilizes H2A-containing and H2A.Z-containing histones equally. Nucleosome mobilization assay was performed as described (Smith and Peterson, 2005). Mononucleosomes were reconstituted with recombinant yeast histone octamers and [α-32P]dCTP-labeled 343 bp DNA fragments the containing 601 sequence located at on end of the DNA.
(C) ATPase activities of INO80 and SWR-C. ATPase activity was measured as described (Smith and Peterson, 2005). Plasmid DNA (1 μg) was used as a substrate. SWR-C showed ∼35% the activity of INO80, consistent with a previous study (Mizuguchi et al., 2004).
(D) INO80 remodels nucleosomal arrays. Restriction enzymes accessibility assay for nucleosomal arrays was performed as described (Logie and Peterson, 1999). Nucleosomal arrays were reconstituted with recombinant Xenopus histone octamers and [α-32P]dCTP-labeled DNA fragments containing 11 repeats of a 208 bp 5S rDNA sequence. SWR-C was inactive in this remodeling assay.
(E) Histone-exchange assays. Exchange assays were performed as in Figure 3, using mononucleosome substrate assembled with FLAG-H2A and free HA-H2A.Z/H2B dimers. Assays demonstrate ATP-dependent incorporation of H2A.Z by SWR-C and eviction of H2A. Note lack of both activities by INO80.
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13. Figure S4
Reduced H2A.Z Expression Suppresses the Replication Defects of the ino80 Mutant Strain, Related to Figure 4
(A) The ino80 htz1 double mutant strain is synthetically lethal. Cells from the ino80 htz1 double mutant strain expressing INO80 from a URA3 plasmid were plated in ten-fold serial dilution on plates lacking uracil or containing 5-FOA to select for cells that have lost the URA3 plasmid. Pictures were taken after either 2 days incubation at 30°C for the SD-URA plates or 6 days for the 5-FOA plates.
(B) Reduced incorporation of HTZ1cp at the KAR4 promoter. Mononucleosomal-ChIP assay for H2A.Z was conducted in G2/M-arrested cells expressing HTZ1 either from the full (HTZ1) or the truncated (HTZ1cp) HTZ1 promoter. Values were measured as in Figure 2G.
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14. Figure S5
The hda1 Mutant Strain Is Sensitive to DNA Damage, Related to Figure 5
WT, arp8, and hda1 mutant cells were plated in 10-fold serial dilutions on YPD plates containing the indicated concentration of zeocin to induce DNA DSBs. Pictures of the plates were taken after 2–5 days incubation at 30°C.
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15. Figure S6
The Genomic Instability Phenotypes Caused by Disruption of the INO80 Complex Are Rescued by the H2A.Z-K(3,8,10,14)Q Acetylation Mimic Mutant, Related to Figure 6
(A) Expression of the HTZ1-K(3,8,10,14)Q allele stabilizes DNA polymerase alpha at the replication fork in the arp8 mutant during replication stress. ChIP analysis was conducted in the indicated strains bearing a myc-tagged POL1 allele, using polyclonal anti-myc antiserum. G1-arrested cells were released into 0.2 M HU and samples were collected at 60' in HU. Occupancy at ARS607 and at sites 1.2 kb and 2.0 kb proximal to the origin (left) and at the late-fired origin, ARS501, (right) was measured by quantitative PCR. Values reflect the fold enrichment of the tested DNA relative to a region of the ASI1 ORF after correction for the ratios of amplification achieved using input DNA. Error bars represent s.d. between two different experiments.
(B) Expression of Htz1 4KQ in the presence of INO80. Left: show 10-fold serial dilutions of WT and HTZ1 4KQ strains on either a YPD plate or a YPD plate containing the indicated concentration of zeocin. Note that this htz1 allele has no apparent phenotype in the presence of INO80. Right: shows that the Htz1 4KQ derivative is expressed normally. Acid-extracted proteins from cells expressing WT H2A.Z (WT) and H2A.Z-K(3,8,10,14)Q [4(K-Q)] were separated by SDS-PAGE and assayed for total H2A.Z and H2A.Z K14ac levels by Western blotting. Equal gel loading was confirmed by immunoblot analysis against Eaf3.
(C) Expression of the HTZ1-K(3,8,10,14)Q allele does not rescue the genome instability phenotypes of the mre11 mutant. WT, mre11 and mre11 cells expressing the HTZ1-K(3,8,10,14)Q allele (HTZ1 4KQ,mre11) were plated in ten-fold serial dilutions on YPD plates containing the indicated concentration of methylmethanesulfonate (MMS) and hydroxyurea (HU). Pictures of the plates were taken after 2-4 days incubation at 30°C.
(D and E) Similar aberrant chromatin incorporation of H2A.Z and H2A.Z-K(3,8,10,14)Q mutant in the arp8 mutant. Chromatin incorporation of H2A.Z and H2A.Z-K(3,8,10,14)Q as measured by ChIP assay. ChIP analysis of H2A.Z was conducted in G1-arrested cells from the indicated strains. Occupancy of H2A.Z at the indicated DNA domains was measured by qPCR. Values reflect the fold enrichment of the tested DNA relative to a subtelomeric region of chromosome VI (coordinates: to  bp) after correction for the ratios of amplification achieved using input DNA. Error bars represent variation between two different experiments, however there was a similar trend in H2A. enrichment between WT and arp8 and WT and Htz1-4K- > Q arp8 samples were similar between experiments.
(F) INO80 can catalyze in vitro dimer exchange with a mononucleosome substrate harboring H2A.Z 4K- > Q. Exchange assays were performed as in Figure 3C.
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16. Figure S7
Antibody Specificity for H2A.Z, Related to Experimental Procedures
(A) Acid-extracted proteins from logarithmically grown WT cells and cells deleted for HTZ1 (htz1) were separated by SDS-PAGE and immunoblot analysis was performed using antibodies against H2A.Z from Active Motif (left) and Millipore (right). Asterisk indicates a cross-reaction of the antibody with a nonhistone protein and serves as equal loading control. Neither of the antibodies recognizes recombinant H2A (data not shown and Altaf et al., 2010). The lower band appearing on the immunoblot for the Millipore H2A.Z antibody does not comigrate with H2A.
(B) Chromatin Immunoprecipitation analysis of H2A.Z was conducted in the indicated strains using antibodies against H2A.Z from Active Motif and Millipore. Values on the top reflect the absolute enrichment of the tested DNA after correction for the ratios of amplification achieved using input DNA. Values on the bottom reflect the fold enrichment of the tested DNA in the WT strain relative to the enrichment in the htz1 mutant strain after correction for the ratios of amplification achieved using input DNA.
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