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DOI: 10.1038/ncb2017 Figure S1 53BP1 sirna results in a heterochromatic DSB repair defect. Panel A: NIH3T3 cells were transfected with murine 53BP1 sirna.

1 DOI: /ncb2017 Figure S1 53BP1 sirna results in a heterochromatic DSB repair defect. Panel A: NIH3T3 cells were transfected with murine 53BP1 sirna. 48 hrs later, cells were irradiated with 3 Gy IR, harvested 24 hr later and immunostained for γh2ax (green) and 53BP1 (red) and DAPI (blue). Representative cells with good or poor 53BP1 knockdown are indicated by arrows. Cells depleted for 53BP1 have persistent γh2ax, the majority of which (60-70%) overlap with the periphery of chromocentres. Panel B: High resolution, deconvolved Z-stacked images were obtained, showing DAPI-chromocentres (blue), γh2ax foci (green) and isolated regions of strong blue-green overlap (red). Using softworx Suite software, quantitative measurements of blue-green overlap were made. Panel C: WT (ATM +/+ ), 53BP1 -/- or MDC1 -/- MEFs were irradiated with 80 Gy and examined 1, 2 or 3 days post IR by PFGE as described in 8. Numbers indicate the fraction of DNA released from the gel plug into the gel (the FAR value ± standard error) and was quantified by evaluating the ethidium bromide-stained PFGE gels. 1

2 Figure S2 Mediator loss or depletion impairs 53BP1 recruitment to IRIF and KAP-1 dependent DSB repair. All scale bars indicate 10 µm. Panel A: 1BR3 primary human fibroblasts were transfected with mock, MDC1, RNF8 or 53BP1 sirna duplexes. 72 hr later, cells were treated with DMSO or ATMi, irradiated with 0.5 Gy IR and harvested 0.5 hr later. Cells were fixed and stained for 53BP1 (red), γh2ax (green) and DAPI (blue). Panel B: 1BR3 cells were transfected as in (A), as indicated, and stained for 53BP1 and MDC1. The relative signal intensity of each was assessed using ImageJ software. Panel C: RIDDLE syndrome fibroblasts stably complemented with HA-vector or HA-RNF168 were irradiated with 3 Gy, harvested as indicated and immunostained as in (A). γh2ax foci were enumerated as in Figure 1. Panel D, E: RIDDLE syndrome fibroblasts as in (C) were transfected with sirna as indicated and treated/scored as in (C). Panel F: A549 cells, transfected as in Figure 1E, were stained for 53BP1 and MDC1 and quantified as in (B). Panel G: The samples in (A) were additionally irradiated with 3 Gy IR, harvested 0.5 hr later, fixed and stained for pkap-1 (red), γh2ax (green) and DAPI (blue). Panel H: In a parallel experiment to Figure 2B, 1BR3 cells were treated with 0-32 Gy IR and harvested 0.5hr later. Cells were trypsinized, washed in PBS and then extracted with PBS containing 0.2% (v/v) Triton X100 for 5 minutes on ice. The resulting pellet was solublized as outlined in the methods for chromatin retention assay. Samples were immunoblotted for the indicated proteins. 2

Figure S3 The anti-pkap-1 antibody exclusively detects pkap-1 by immunoblot, immunoprecipitation and immunofluorescence. All scale bars indicate 10 µm.

3 Figure S3 The anti-pkap-1 antibody exclusively detects pkap-1 by immunoblot, immunoprecipitation and immunofluorescence. All scale bars indicate 10 µm. Panel A: The KAP-1 phosphospecific antibody was conjugated to Protein-A Sepharose (PAS) beads. pspecific conjugated or non-conjugated beads (mock) were incubated with 2 mg of whole cell extract prepared from GM02188 wildtype LBLs irradiated with or without 20 Gy IR. 4 hr later, beads were exhaustively washed with lysis buffer and then boiled in SDS sample buffer. Immunoprecipitates were divided between an immunoblot for the pkap-1 antibody (upper panel) or Coomassie stain gels (lower panel). Panel B: Coomassie stain bands were identified by massspectrometry to be human KAP-1. Peptides identified in the trypsin-digested IP are indicated in red. The underlined sequence (LSSQE) corresponds to the peptide region that the p-specific antibody recognized (phosphoserine 824 of KAP-1). Panel C: GM02188 cells were irradiated with 40 Gy IR and harvested as indicated. Whole cell extracts were prepared and immunoblotted for pkap-1. Panel D: GM02188 cells were irradiated with 2 Gy IR and harvested 0.5 hr later. Whole cell extracts were prepared and incubated with un-conjugated total KAP-1 antibody or PAS-conjugated pkap-1 antibody. Washed immunoprecipitates were immunoblotted for pkap-1 and total KAP1. The asterisk indicates background signal resulting from immunoblotting with the same antibody as used for IP (N.B. this was markedly reduced in Figures 3 and 5 by increasing the cross-linking time used to prepare the conjugated antibody used for IP) Panel E: A549 cells were transfected with mock or KAP-1 sirna. 48 hr post transfection, cells were treated with or without ATMi and irradiated with 2 Gy IR and harvested 0.5 hr later. Whole cell extracts were prepared and immunoblotted for pkap-1, total KAP-1 and Ku70. Panel F: Stationary-phase 1BR3 (normal) primary human fibroblasts, 1BR3 cells pre-incubated with ATMi or AT1BR (ATM mutant) primary human fibroblasts were irradiated with 3 Gy IR and harvested at the indicated times. Cells were fixed and immunostained for pkap-1 (red), γh2ax (green) and DAPI (blue). Panel G: NIH3T3 cells were transfected ± pegfp vectors encoding WT or S824A human KAP-1 cdna. 16 hr later, cells were irradiated with 1 Gy, harvested 0.5 hr later and stained with pkap-1 (red) and DAPI (blue). Green signal indicates GFP. Anti-pKAP-1 does not detect murine KAP-1 by IF. pkap-1 was detected (and enriched at HC chromocenters) in NIH3T3 cells transfected with WT but not S824A human KAP-1 (N.B. Further analysis of pkap-1 in NIH3T3 cells using transfected human KAP-1 was precluded by the toxicity of over-expressed human KAP-1 to murine cells over time). 3

4 Figure S4 Analyzing pkap-1 foci. All scale bars indicate 10 µm. Panels A,B: Confluence-arrested RIDDLE Syndrome human fibroblasts complemented with RNF168-HA or HA alone were irradiated and stained as indicated. Panel C: A sample image (the 4hr time point of Figure 2A) showing γh2ax foci with (i) or without (ii) strong pkap-1 signal; ImageJ software tools measure the relative green and red intensity within each outlined area and generate quantitative values that are plotted as shown in (D). Panel D: 1BR3 (normal) and 2BN (XLF mutant) primary human fibroblasts were irradiated and harvested as indicated. Cells were fixed and stained with pkap-1 (red), γh2ax (green) and DAPI (blue). Using ImageJ software, the intensity of γh2ax and pkap-1 signal at the site of γh2ax foci were quantified. pkap-1 signal at regions with no γh2ax was separately measured to quantify the average pan-nuclear background pkap-1 signal. The relative pkap-1 and γh2ax level for each analyzed foci was plotted for each cell type/condition as indicated. The average pkap-1 signal throughout the nucleus was ~60 AU (arbitrary units) ± 20. To be classified as being associated with a pkap-1 foci, a given foci of γh2ax must have an overlapping pkap-1 level greater than ~80, i.e. greater than the highest standard deviation of the average pan-nuclear background pkap-1 signal. The absolute numbers of foci with pkap-1 levels over ~80 AU were scored relative to the total number of foci to generate the percentages shown in figure 2D. 4

5 Figure S5 Controls for 53BP1 expression and MRN-dependent DSB repair. All scale bars indicate 10 µm. Panel A: HeLa cells were transfected with 1 µg of pcmh6k-53bp1 FL and, 16 hr later, irradiated and stained as indicated. Note the dramatic increase in 53BP1 signal but not patm signal in cells over-expressing HA-53BP1 FL. Panel B: Immunofluorescent HA, 53BP1 and patm signal was quantified by ImageJ software for cells expressing the indicated constructs. Expression of HA-53BP1 FL significantly increased the average signal detected by the 53BP1 antibody. No significant increase in patm signal was observed following HA-53BP1 FL expression, strongly suggesting that patm (rabbit monoclonal from Epitomics, Inc.) does not cross-react with 53BP1. Panel C: Confluence-arrested 1BR3 (normal), ATLD2 (Mre11 mutant) and CZD82Ch (NBS1 mutant) primary human fibroblasts were irradiated with 3 Gy IR and harvested as indicated. Cells were stained for pkap-1 (red), γh2ax (green) and DAPI (blue). Panel D: γh2ax foci were enumerated for the experiment described in (C), with the inclusion of 24 hr time points from cells additionally treated with ATMi for the duration of the experiment. Panel E: 1BR3 (normal) and ATLD2 (Mre11 mutant) were transfected ± KAP-1 sirna and irradiated/harvested as in (C). Cells were stained for γh2ax and KAP-1 and γh2ax foci numbers were enumerated in cells with and without KAP-1 knockdown. 5

6 Figure S6 A model for 53BP1-dependent HC DSB repair. (i) DSB induction triggers ATM activation; (ii) ATM activity spreads throughout the nucleus and phosphorylates KAP-1 diffusely, insufficient for DSB repair at KAP-1 rich HC; (iii) ATM activity is concentrated at the DSB via 53BP1, enabling robust, local pkap-1 and facilitating HC DSB repair. 6

Figure S7 Full-size scans of immunoblots. Panel A: Uncropped blots for figure 1F. Panel B: Uncropped blots for figure 3E. Panel C: Uncropped blots for figure 4D.

7 Figure S7 Full-size scans of immunoblots. Panel A: Uncropped blots for figure 1F. Panel B: Uncropped blots for figure 3E. Panel C: Uncropped blots for figure 4D. All numbers denote approximate position and size (in kda) of molecular weight markers. Times indicated on the immunoblots refer to the length of exposure to film. Blots shown in a singular box were exposed to same piece of film for same time. 7

Figure S8 Full-size scans of immunoblots. Panel A: Uncropped blots for figure 4E. Panel B: Uncropped blots for figure S2H. Panel C: Uncropped blots for figure S3A.

8 Figure S8 Full-size scans of immunoblots. Panel A: Uncropped blots for figure 4E. Panel B: Uncropped blots for figure S2H. Panel C: Uncropped blots for figure S3A. Panel D: Uncropped blots for figure S3D. Panel E: Uncropped blots for figure S3E. All numbers denote approximate position and size (in kda) of molecular weight markers. Times indicated on the immunoblots refer to the length of exposure to film. Blots shown in a singular box were exposed to same piece of film for same time. 8

9 Supplementary Discussion Previously, dominant-negative 53BP1, but not MDC1, expression was shown to suppress NHEJ in I-SceI based assays 1, suggesting an MDC1-independent role of 53BP1 in NHEJ. MDC1 or H2AX loss also had a smaller impact on long range V(D)J recombination than 53BP1 2. Here, we demonstrate that MDC1 and 53BP1 function within the same pathway to repair IR-induced DSBs. The discrepancy could be explained by differences in repairing enzymatically-induced breaks in reporter constructs (or at CSR loci) versus radiation-induced lesions in chromatin. It is also possible that cell cycle differences (our studies are in G0/G1) or distinct effects of dominant-negative over-expression versus endogenous protein depletion (on the interplay between MDC1 and 53BP1) underlie the differing MDC1-dependency between our studies. In the absence of MDC1 or H2AX, 53BP1 is transiently recruited but not retained at DSBs 3 and so it is possible that transient 53BP1 accumulation may suffice for some endpoints. Our studies suggest that HC DSB repair requires prolonged ATM activation at the DSB, thus MDC1 and H2AX, which promote prolonged 53BP1 retention at DSBs, are essential. The consequences of KAP-1 phosphorylation on HC are uncertain. There has been some suggestion that pkap-1 impinges upon the SUMOylation of KAP-1 s bromodomain, an event that is required for KAP-1 mediated gene repression 4,5. The SUMOylation of KAP-1 is pre-requisite for the recruitment of the SETDB1 histone methyltransferase and the ATP-dependent chromatin remodeling enzyme Mi-2α/CHD3 6, 7, a component of the NuRD (Nucleosome Remodelling and Deactylase) complex. Whether KAP-1 phosphorylation induced loss of SUMOylation has a meaningful impact upon either KAP-1 dependent SETDB1 or NuRD recruitment (or activity) at the DSB site has yet to be determined. 1

10 Supplementary References 1. Xie, A. et al. Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair. Mol Cell 28, (2007). 2. Difilippantonio, S. et al. 53BP1 facilitates long-range DNA end-joining during V(D)J recombination. Nature 456, (2008). 3. Yuan, J. & Chen, J. MRE11/RAD50/NBS1 complex dictates DNA repair independent of H2AX. J Biol Chem (2009). 4. Li, X. et al. Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression. J Biol Chem 282, (2007). 5. Lee, Y.K., Thomas, S.N., Yang, A.J. & Ann, D.K. Doxorubicin down-regulates Kruppel-associated box domain-associated protein 1 sumoylation that relieves its transcription repression on p21waf1/cip1 in breast cancer MCF-7 cells. J Biol Chem 282, (2007). 6. Schultz, D.C., Friedman, J.R. & Rauscher, F.J., 3rd Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD. Genes Dev 15, (2001). 7. Schultz, D.C., Ayyanathan, K., Negorev, D., Maul, G.G. & Rauscher, F.J., 3rd SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 16, (2002). 8. Goodarzi, A.A. et al. ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol Cell 31, (2008). 2

Supplementary Discussion and Figures

Supplementary Discussion and Figures Differences in chromatin retention between the two CHD3 isoforms The pan-nuclear loss of the KAP-1-interacting CHD3 isoform from chromatin (observed in Fig. 2A) is