Aaron A. Goodarzi, Angela T. Noon, Dorothee Deckbar, Yael Ziv, Yosef Shiloh, Markus Löbrich, and Penny A. Jeggo

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1 Molecular Cell, Volume 31 Supplemental Data 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, and Penny A. Jeggo Supplemental Experimental Procedures Reagents and Irradiation: The ATM inhibitor (ATMi) KU was a gift from KuDos Pharmaceuticals and was used at a final concentration of 10 µm. Micrococcal nuclease (MNase) and neocarzinostatin were obtained from Sigma-Aldrich (Poole, UK). Calicheamicin was a provided by Ola Hammarsten. Irradiation with low energy (278 ev) Carbon-K characteristic X-rays was performed as previously described (Kuhne et al., 2005). Immunofluorescence Cells plated on glass slides were fixed for 10 min with fixative (3% (w/v) PFA, 2% (w/v) sucrose, 1X PBS) and permeablized for 1 min with 0.2 % Triton X-100 in PBS. Cells were rinsed with PBS and incubated with primary antibody diluted in PBS + 2% (w/v) BSA for 1 hour at room temperature (RT). Cells were washed three times, incubated with secondary antibody (diluted in PBS + 2% (w/v) BSA) for 30 min at RT in the dark, incubated with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and washed three times with PBS. Slides were mounted using Vectasheild and visualized using a Zeiss Axioplan microscope and Simple-PCI software. For high resolution 3D imaging of deconvolved Z-stacks, slides were analyzed using an Applied Precision DeltaVision RT Olympus IX70 deconvolution microscope and softworx Suite software. Antibodies and Immunoblotting The following antibodies were used at indicated concentrations for immunofluorescence: Anti-KAP-1 ab10484 (1:1000, Abcam, UK), anti-γh2ax (1:800, Upstate Technology, UK), anti-tri-me K9 Histone H3 ab8898 (1:800, Abcam, UK), anti- CENP-A M-85 (1:100, Santa Cruz, CA), anti-hp1 (non isoform-specific antibody, i.e. detects HP1 α, β and γ) Fl-191 (1:100, Santa Cruz, CA, CA), anti-hdac1 ab19895 (1:300, Abcam, UK), anti-hdac2 H-54 (1:300, Santa Cruz, CA), 53BP1 ab21083 (1:800, Abcam UK), acetyl K9 histone H3 ab12179 (1:1000, Abcam UK), anti-hmg1 (1:400, Pharmingen, CA), anti-e2f1 E-20 (1:800, Santa Cruz, CA), anti-ku70 M19 (1:100, Santa Cruz, CA). Appropriate secondary antibodies were used at a 1:200 (green = FITC; red = Cy3 or TRITC conjugated antibodies). For immunoblotting, clarified

2 extracts were resolved on 7.5% denaturing PAGE and transferred to nitrocellulose (100V, 60 minutes in 48 mm Tris-base, 39 mm glycine and 20% (v/v) methanol). Chromosomal break analysis Confluent GM00157 cells were treated with 20 µm ATMi 30 min, then irradiated with 2 Gy, incubated for 24 hours and sub-cultured in the presence of ATMi for a further 32 hours. At this time point, maximum numbers of G2 phase cells were obtained as determined by flow cytometry. 50 ng/ml Calyculin A was added 30 minutes 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 analyzed by FISH using probes against chromosomes 7, 8 and X, according to the manufacturer s instructions (Metasystems, Germany). Semi-automated microscopy was performed using a Zeiss Axioplan2 microscope and Metafer and ISIS software (Metasystems, Germany). Expression of GFP-KAP-1 in cells with KAP-1 knockdown 1BRneo cells were transfected with 100 pmol KAP-1 B sirna per 2 x 10 5 cells, using Metafectene -Pro (according to manufacturer s instructions). 48 hr later, cells were trypsinized and simultaneously transfected with both 100 pmol KAP-1 B sirna and 1 µg pegfp-kap1-wt, S824A or S824D (or an equivalent volume of water) per 2 x 10 5 cells using Metafectene -Pro as before. 2 x10 5 transfected cells (per condition) were then plated onto glass slides in 2 ml media. 16 hours later, cells were treated ± ATMi and irradiated as normal. Nucleosome Size Control 1 x 10 7 cells were washed with PBS and once with LSB. Pelleted cells were resuspended in 5X PCV of LSB µm MC-LR and 1X protease inhibitor cocktail (Sigma-Aldrich, UK) and snap-frozen in liquid nitrogen. Cells were quick-thawed and immediately centrifuged for 10 min at rpm. The resulting supernatant was discarded and the pellet resuspended in 2X the original PCV of nuclease buffer containing 100 U/mL MNase. Samples were incubated at 37 o C for 0, 5, 10, 20, 40 or 80 min before addition of an equal volume of denaturing buffer and storage on ice. When all samples were collected, an equal volume of TE buffer was added followed by 1.5 V of 1:1 Phenol:Chloroform. Samples were inverted five times and centrifuged rpm for 10 min. The phenol (lower) phase was then removed, followed by the addition of Acetic Acid to 450 mm and then 7.5 V of ice-cold Ethanol containing 15 µg/ml of glycogen. Samples were inverted, stored on ice 5 minutes and centrifuged rpm for 10 min. The supernatant was then discarded and the DNA pellet washed with 0.5 ml 70% Ethanol. Pellets were air-dried for 10 min before being resuspended in 100 µl water. 1.5 µg DNA was resolved on a 2% (w/v) agarose gel and visualized by Ethidium Bromide staining. Pulsed Field Gel Electrophoresis (PFGE) For PFGE analysis, WT or ATM -/- MEFs were irradiated 48 hr after transfection with 30 nm mouse KAP-1 C sirna or mouse ATM sirna (Qiagen) using Metafectene -Pro. Transfection efficiency was greater than 75% (assessed under the microscope by the 2

number of cells showing loss of KAP1 immunofluorescent signal). At the time of irradiation, cultures were nearly confluent (>80% in G1, assessed by flow cytometry).

3 number of cells showing loss of KAP1 immunofluorescent signal). At the time of irradiation, cultures were nearly confluent (>80% in G1, assessed by flow cytometry). PFGE analysis was performed according to Kühne et al (Cancer Res 64, ) with slight modifications. In short, cells were harvested either immediately after irradiation with 10 Gy (to assess DSB induction) or at 24 and 48 hr after irradiation with 80 Gy (to assess DSB repair). Cells irradiated to assess DSB induction were kept on ice 10 min before and during the irradiation procedure. Cells were then harvested on ice, swiftly embedded into 0.75% agarose plugs and immediately transferred back to 0 C for 1 h. Cells were then lysed at 50 C for 24 hr. The fraction of DNA released from the gel plug into the gel (the FAR value) was quantified by evaluating the ethidium bromidestained PFGE gel (Figure S8C). In contrast to previous studies involving less stringent cooling conditions to assess DSB induction (Deckbar et al., 2007, J Cell Biol 176, ), higher FAR values were obtained for the 10 Gy induction samples in this study (Figure 5F,G). This may have been the result of the generation of heat-labile sites during irradiation which are converted into DSBs during the lysis step of PFGE and which can lead to an over-estimation of the true DSB induction value by a factor of nearly two (Rydberg, 2000, Radiat Res 153, ). Figure S1: A maximum of ~25% of DSBs induced by NCS require ATM-signalling for repair. 1BR3 (wildtype) and AT1BR (ATM-/-) primary cells were treated with either 2 Gy IR or 50 ng/µl NCS. Cells were fixed at the indicated times post treatment and stained for H2AX (green) and DAPI (blue). 3

4 Figure S2: γh2ax foci remaining un-repaired in cells treated with ATMi are visibly associated with heterochromatin. Panel A: DMSO or 10 µm KU55933 ATM inhibitor (ATMi) was added to confluent, stationary-phase NIH 3T3 cells and, 30 minutes later, cells were irradiated with 2 Gy IR and harvested 0.5, 2, 6, 16 and 24 hours post-ir. Cells were fixed and immunostained for γh2ax (red) and DAPI (green). All images shown are overlays and are representative of the total cell population. Panel B: NIH3T3 cells were treated with ATMi, irradiated with 3 Gy IR, harvested 24 hr later and immunostained for 53BP1 (red), γh2ax (green) and DAPI (blue). Panel C: Numbers of 53BP1 foci were scored for NIH3T3 cells treated ± ATMi, irradiated with 3 Gy IR and harvested as indicated. Panel D: ATM -/- MEFs were irradiated with 3 Gy IR and harvested 24 hr later. Cells were immunostained with TriMe K9 H3 (red), γh2ax (green) and DAPI (blue). A high contrast image of the indicated cell is shown, with arrows pointing to foci scored as heterochromatic. 4

5 Figure S3: High resolution imaging of γh2ax foci remaining un-repaired in cells treated with ATMi. Panel A: NIH3T3 cells were treated with ATMi, irradiated with 3 Gy IR and harvested 24 hr later. Cells were stained with γh2ax (green) and DAPI (blue). High resolution Z-stack images were captured and deconvolved using the softworx Suite software. Regions of intense DAPI staining (purple outlines) and γh2ax foci (green outlines) were isolated for the whole stack. A single plane of view is shown. The right panel shows a close-up of the region outlined in white. Regions of isolated overlap can clearly be seen (indicated by white arrows), where γh2ax spreads into the surface of the DAPI chromocenters. Panel B: Signal intensity chromatographs are shown for three separate slices through the indicated nucleus. Green lines indicate γh2ax signal, blue lines indicate DAPI signal. Light blue dashed lines indicate the DAPI signal thresholds of euchromatin and heterochromatin. 5

6 6

7 Figure S4: >70% of γh2ax foci remaining un-repaired in cells treated with ATMi are visibly associated with heterochromatin. Panel A: DMSO or ATMi was added to stationary-phase NIH 3T3 cells and, 30 minutes later, cells were irradiated with 0.25, 0.5, 0.75, 1 or 2 Gy IR and harvested 0.5 hour later. Cells were fixed and immunostained for γh2ax (red) and DAPI (green). All images shown are overlays and are representative of the total cell population (there was no difference between DMSO and ATMi at this time point). The total number of γh2ax foci and γh2ax foci associated with regions of heterochromatin were counted for a minimum of 20 cells and the average of three experiments is shown. Panel B: DMSO or ATMi was added to stationary-phase NIH 3T3 cells and, 30 minutes later, cells were irradiated with 1, 2 or 3 Gy IR and harvested 24 hours later. Cells were fixed, immunostained and counted as above. 7

Figure S5: DNA size control for nucleosome preparations. Left Panel: Untreated NIH3T3 cells were processed for nucleosome solublization as described in the Experimental Procedures.

8 Figure S5: DNA size control for nucleosome preparations. Left Panel: Untreated NIH3T3 cells were processed for nucleosome solublization as described in the Experimental Procedures. Samples were incubated for the indicated times with 100 U/mL MNase, then extracted with Phenol:Chloroform and ethanol precipitated to isolate DNA. 1.5 µg of DNA was loaded on a 2% (w/v) agarose gel and visualized by Ethidium Bromide staining. Right Panel: NIH3T3 cells were processed for chromatin segregation as described in the Experimental Procedures. Samples at each stage of the segregation were extracted with Phenol:Chloroform and ethanol precipitated to isolate DNA. 1.5 µg of DNA was loaded on a 2% (w/v) agarose gel and visualized by Ethidium Bromide staining. 8

9 Figure S6: ATM-phosphorylation site mutants of KAP-1 affect the ATM-dependent DSB repair defect of 1BRneo cells. 1BRneo cells were transfected with sirna duplexes to human KAP-1 (KAP-1 B ) or scrambled sirna (mock). 48 hr later, cells were re-transfected with sirna with or without pegfp vectors encoding wildtype, S824A or S824D human KAP-1 sirna-resistant cdna. 24 hr later, cells were treated ±ATMi, irradiated and harvested as indicated. Cells were fixed and stained for 53BP1 (red) and DAPI (blue). GFP expression = green. 9

10 Figure S7: Knockdown of heterochromatin building factors in 1BRneo cells using sirna oligos. 2.3 x 105 1BRneo cells were transfected with either scrambled sirna (mock) or 20 µmol of sirna directed towards either KAP-1 (panel A), HP1α, β & γ isoform pan-specific antibody (panel B) or HDAC1 and HDAC2 (panel C). Cells were harvested and fixed 48 or 72 hours relative to the transfection procedure, as indicated. Fixed cells were immunostained for KAP-1 (panel A, red), HP1αβγ (panel B, red) or HDAC1 and HDAC2 (panel C, red). All cells were counterstained with DAPI (blue). Overlaid images (overlay signal, purple) appear in the top left-hand corner of each image. 10

Figure S8: KAP-1 sirna does not affect DAPI chromocenter integrity in NIH3T3 cells, but does alter histone H3 trimethylation and alleviates the requirement for ATM in DSB assessed by PFGE.

11 Figure S8: KAP-1 sirna does not affect DAPI chromocenter integrity in NIH3T3 cells, but does alter histone H3 trimethylation and alleviates the requirement for ATM in DSB assessed by PFGE. Panel AB: NIH3T3 cells were transfected with scrambled control sirna (Mock) or 100 pmol KAP-1 C sirna using Metafectene-Pro. 48 hrs post transfection, cells were harvested and stained for either KAP-1 (red, Panel A) or TriMe K9 H3 (red, Panel B). Panel C: Representative pulsed-field gels for WT or ATM -/- MEFs transfected with either ATM or KAP-1 sirna and irradiated as described in the Experimental Procedures. 11

Evaluation of Metafectene-Pro mediated transfection of sirna and/or plasmids into human and murine cells

Evaluation of Metafectene-Pro mediated transfection of sirna and/or plasmids into human and murine cells Aaron A. Goodarzi and Penny A. Jeggo Genome Damage and Stability Centre, University of Sussex, East