1. Volume 31, Issue 2, Pages 167-177 (July 2008)
ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin 
Aaron A. Goodarzi, Angela T. Noon, Dorothee Deckbar, Yael Ziv, Yosef Shiloh, Markus Löbrich, Penny A. Jeggo 
Molecular Cell 
Volume 31, Issue 2, Pages (July 2008)
DOI: /j.molcel
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2. Figure 1 A Maximum of ∼25% of DSBs Require ATM Signaling for Repair
(A) 1BR3 (WT) and AT1BR (ATM−/−) primary human fibroblasts were irradiated (or not) with 2 Gy (γ-ray) IR.
(B) 1BR3 (WT) and AT1BR (ATM−/−) primary human fibroblasts were treated with 50 ng/ml NCS.
(C) HSF3 (WT) or AT1BR (ATM−/−) primary human fibroblasts were irradiated (or not) with 1 Gy of carbon-K X-rays. Here and in all subsequent figures, where indicated, cells were fixed at the indicated times, immunostained for γH2AX, and foci were enumerated for >30 cells per condition (blind counting). The asterisk in (C) indicates that we were unable to count the 0.25 hr time point in the A-T cell, due to delayed foci formation. All results represent the mean ± SD of three experiments.
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3. Figure 2
γH2AX Foci Remaining in ATMi-Treated Cells Are Associated with Regions of Heterochromatin
(A) Untreated NIH 3T3 cells were fixed and stained for trimethylated K9 of histone H3, CENP-A, KAP-1, HP1αβγ, HMGB1, or E2F1, as indicated (red), before being counterstained with DAPI (blue).
(B) DMSO or 10 μM KU55933 (ATMi) was added to confluent, stationary-phase NIH 3T3 cells and, 0.5 hr later, cells were irradiated with 2 Gy IR and harvested 24 hr post-IR. Cells were fixed and stained for H2AX (green), trimethylated K9 of histone H3 (red), and DAPI (blue). All images shown are representative of the total cell population.
(C) ATMi-treated NIH 3T3 cells were irradiated with 3 Gy IR and harvested 24 hr later. Cells were fixed and stained for γH2AX (green) and DAPI (blue). High-resolution Z stacks of deconvolved images were acquired for a representative cell demonstrating the ATM-dependent repair defect (Ci–Civ). softWoRx was used to isolate γH2AX foci (Cii) and intensely staining DAPI chromocenters (Ciii), and the regions of overlap between them was assessed (Civ, colored peach in [Cii]–[Civ]). The regions isolated in (Cii)–(Civ) were used to generate three-dimensional models of γH2AX foci (green) and heterochromatic chromocenters (blue) (Cv–Cx). (Cvi)–(Cx) are rotations of the image shown in (Cv).
(D) NIH 3T3 cells were treated as in (B) and harvested at the indicated time points. Total γH2AX foci numbers were scored.
(E) γH2AX foci juxtaposed with the heterochromatic regions (assessed by DAPI staining) were also scored.
(F) The data generated in (C) and (D) were used to estimate the percent remaining γH2AX foci associated with euchromatin (EC, green circles) or heterochromatin (HC, red squares), in the presence of DMSO (filled circles/squares) or ATMi (open circles/squares). All results represent the mean ± SD of three experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
DNA Double-Strand Breaks Dependent upon ATM Activity for Repair Are Associated with Regions of Heterochromatin
(A) Confluent GM00157 (Klinefelter syndrome) cells were treated with or without ATMi, irradiated with 2 Gy IR, incubated 24 hr, subcultured (±ATMi), and harvested after an additional 32 hr (when the maximum number of G2-phase cells was obtained as determined by flow cytometry). A total of 50 ng/ml calyculin A was added 0.5 hr prior to harvest to induce premature chromosome condensation of G2 cells. Fixation and preparation of chromosome spreads was as described previously (Deckbar et al., 2007). Slides were processed for FISH against chromosomes 7, 8, and X, and the numbers of chromosomes breaks per metaphase were scored.
(B) Stationary-phase Suv39H1/2 double-knockout or WT MEFs were treated with or without ATMi, irradiated with 3 Gy, and harvested as indicated. Cells were fixed, stained for γH2AX and enumerated.
(C) 48BR (WT) and AG11513 (HGPS) primary human fibroblasts were treated as in (B).
(D) NIH 3T3 cells were treated with or without ATMi, irradiated, and harvested as indicated. Where ATMi was used, the drug was removed 0.5 hr before harvesting. Cells were lysed and nucleosomes solubilized as described in the Experimental Procedures. Soluble nucleosome extract (60 μg) was blotted for γH2AX and trimethylated K9 of histone H3 (TriMe K9 H3). A Ponceau-S stain of visible histones is shown. Duplicate samples were processed for γH2AX immunofluorescence, and the numbers of γH2AX foci were scored.
(E) NIH 3T3 cells were treated as in (D). γH2AX was immunoprecipitated from 1 mg of soluble nucleosome extract, washed with solubilization buffer, and blotted for γH2AX, TriMe K9 H3, and acetyl K9 H3.
(F) The immunoblot data in (E) were quantified and expressed as percent of the maximal TriMe K9 H3 signal divided by percent of maximal γH2AX signal. All results represent the mean ± SD of three experiments.
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5. Figure 4
Knockdown of KAP1 by shRNA Alleviates the DSB Repair Defect of 1BRneo Cells Treated with ATM Inhibitors
(A) 1BRneo cells were transfected with pRETRO-super vector encoding GFP or KAP-1 shRNA. Cells were fixed and stained for KAP-1 (red) and DAPI (blue).
(B and C) At 24–48 hr posttransfection (as in [A]), cells were treated with or without ATMi, irradiated with 3 Gy IR, harvested and stained for KAP-1 (red), γH2AX (green) and DAPI (blue). In (B), a representative image of an ATMi-treated, irradiated cell 24 hr post-IR stained for KAP-1 (red), γH2AX (green) and DAPI (blue). A cell with KAP-1 knockdown is shown adjacent to an unsuccessfully transfected cell. (C) Representative images of γH2AX and DAPI overlays.
(D) γH2AX foci numbers in KAP-1 knockdown cells were quantified.
(E) 1BRneo cells were transfected with siRNA duplexes to human KAP-1 (KAP-1 “B,” identical sequence to system used in [A]) or scrambled siRNA (mock). After 48 hr, cells were retransfected with siRNA with or without pEGFP vectors encoding WT, S824A, or S824D human KAP-1 siRNA-resistant cDNA. After 24 hr, cells were fixed and stained for KAP-1 (red) and DAPI (blue). GFP expression is shown in green.
(F) 1BRneo cells were transfected as in (E) and treated as in (B) and (C). Cells were fixed and stained with 53BP1 and DAPI. Numbers of 53BP1 foci were enumerated. All results represent the mean ± SD of three experiments.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Knockdown of KAP-1, HP1, or HDAC1 and -2 Alleviates the DSB Repair Defect of ATM-Inhibited Cells
(A) 1BRneo cells were transfected with siRNA duplexes targeted to human KAP-1 (KAP-1 “A” siRNA) or scrambled siRNA (mock). At 48 hr posttransfection, cells were treated with or without ATMi, irradiated with 3 Gy IR, harvested, and stained for KAP-1 and γH2AX. γH2AX foci were enumerated in cells with verified knockdown.
(B) 1BRneo cells were transfected with scrambled siRNA, combined siRNA duplexes targeted to HP1α, HP1β and HP1γ (HP1 siRNA), or HP1 and KAP-1 siRNA as in (A). Cells were treated as in (A) and stained for HP1 and γH2AX. γH2AX foci were enumerated as in (A).
(C) 1BRneo cells were transfected with scrambled siRNA or siRNA duplexes targeted to either HDAC1 or HDAC2, treated as in (A), and stained for H2AX and HDAC1 or HDAC2. H2AX foci were enumerated as in (A).
(D) 1BRneo cells were transfected with scrambled siRNA or siRNA duplexes targeted to HDAC1 and HDAC2 siRNA as in (A). Cells were treated as in (A) and stained for γH2AX and HDAC1 or HDAC2. γH2AX foci were enumerated as in (A).
(E) WT or ATM−/− MEFs were transfected with KAP-1 “C” siRNA or scrambled siRNA (mock) with Metafectene-Pro. At 48 hr posttransfection, cells were treated and enumerated as in (A).
(F) WT MEFs were transfected with scrambled (control) siRNA, ATM siRNA, and/or KAP-1 siRNA. At 48 hr posttransfection, cells were irradiated as indicated and processed for PFGE. FAR values represent the fraction of DNA released from the gel plug.
(G) ATM−/− MEFs were treated as in (F). All results represent the mean ± SD of three experiments.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
KAP-1 Association with Nuclease-Resistant Chromatin Changes after IR
(A) Confluent, stationary-phase NIH 3T3 cells were exposed to 0 or 40 Gy IR and harvested 1 hr later. Cells were processed for chromatin segregation as described in the Experimental Procedures. Samples were blotted for HMGB1, KAP1, HP1, γH2AX, and HDAC1, as indicated. Ponceau-S stains of histones are also shown. Note: while anti-KAP-1 signal decreased in C3 after IR, antibody affinity was not affected by KAP-1 phosphorylation in whole-cell extracts (data not shown). When overexposed, weak KAP-1 signal appeared in the P10 and/or C1 fractions after IR (data not shown).
(B) Confluent, stationary-phase NIH 3T3 cells were exposed to 0–80 Gy IR, as indicated, and harvested 0.5 hr later. Cells treated with 80 Gy IR were additionally harvested at indicated times. Samples were processed as in (A), and the C3 fraction was blotted as in (A).
(C) DMSO or ATMi was added to confluent, stationary-phase NIH 3T3 cells and, 0.5 hr later, cells were irradiated as indicated and harvested 0.5 hr later. Samples were processed and the C3 fraction was blotted as in (A).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions