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Supplemental Information DYRK1A Regulates the Recruitment of 53BP1 to the Sites of DNA Damage in Part Through Interaction with R

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Supplemental Information DYRK1A Regulates the Recruitment of 53BP1 to the Sites of DNA Damage in Part Through Interaction with R Supplemental Information DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169. Vijay R. Menon, Varsha Ananthapadmanabhan, Selene Swanson, Siddharth Saini, Fatmata Sesay, Vasily Yakovlev, Laurence Florens, James A. DeCaprio, Michael P. Washburn, Mikhail Dozmorov, Larisa Litovchick Supplemental Figure Legends Figure S1. Validation the DYRK1A interactions detected by MudPIT. A. Comparison of DYRK1A interacting proteins detected in HEK293T cells (Varjosalo et al., 2013) and T98G cells (this paper). Asterisk indicates the proteins known to interact with adenovirus E1A according to the UniProt database (http://www.uniprot.org/uniprot/P03255#interaction). DYRK1A binds E1A indirectly through DCAF7 (Glenewinkel et al., 2016). B. T98G cells stably expressing Flag-HA tagged DCAF7, LZTS2, TROAP and FAM117B (C-terminal fragment 245-589 a.a.) or GFP (control) were used for endogenous DYRK1A IP followed by WB with anti-HA antibody. C. Reciprocal IP/WB assays confirming the interaction between DYRK1A and the indicated candidate interacting proteins at the endogenous levels in T98G cells. Normal rabbit IgG serves as negative control. Figure S2. Induced expression of DYRK1A inhibits 53BP1 accumulation at DSB sites. A. Representative images (63x) of the indicated inducible U-2 OS cell lines treated with doxycycline for 16h, γ-irradiated (5Gy) and incubated for 3h before staining with anti-53BP1 antibody and DAPI. Figure S3. Characterization of the DYRK1A phospho-site mutants of RNF169. A. Representative images show that RNF169-DD mutant are recruited to IRIFs similar to the wild type RNF169. U-2 OS stably expressing RNF169-DD were γ-irradiated (5Gy) and incubated for 3h before staining with anti-HA antibody to detect RNF169. B-D. Quantitation of the HA-RNF169-DD and 53BP1 IRIF formation in U-2 OS cells shown in A. Graph shows average ± stdev of 3 independent experiments. E. Ubiquitin binding assay reveals no differences between the wild type and the phosphosite-mutant RNF169 proteins. HA-tagged RNF169 constructs were transiently expressed in HEK293T cells, extracted using RIPA buffer, immunoprecipitated with anti-HA antibodies and incubated with K63 poly-ubiquitin chains (Boston Biochem, Cat# UC-330) for 2 h at 4°C. After washing the beads, bound ubiquitin as well as RNF169 proteins in the samples were detected by WB. F. Co- immunoprecipitation assay shown that disruption of the DYRK1A phosphorylation sites in RNF169 does not affect its interaction with USP7. Figure S4. shRNA depletion of DYRK1A inhibits recruitment of RNF169 and 53BP1. A. DYRK1A depletion causes a modest decrease of HA-RNF169 IRIF formation. HA-RNF169 was transiently expressed in control and DYRK1A-depleted U-2 OS cells. Graph shows quantification (average ± stdev, N=3) of HA- RNF169 foci in the control and DYRK1A-depleted cells 3 h after γ-radiation (5 Gy). B. Depletion of DYRK1A using shRNA results in decreased 53BP1 IRIF formation. U-2 OS cells were treated and analyzed as in panel A. Western blots above the graphs confirm the efficiency of shRNA-mediated depletion of DYRK1A. Figure S5. Generation of cell lines devoid of DYRK1A expression using CRISR-Cas9 approach. A. DNA fluorescence in situ hybridization (FISH) assay using two different probes specific to DYRK1A labeled with Cy3 (red) and two control probes labeled with FITC (green) detects a single allele of DYRK1A gene in U-2 OS cell line. Schematic of human ch21 regions spanned by the FISH probes specific to DYRK1A gene (shown in red) and control regions (shown in green), as well as representative cell images are shown. The DNA probes were obtained from the BACPAC Resource Center, the Children's Hospital Oakland Research Institute (Oakland, CA), labeled and used for FISH assay as described in (Zheng et al., 2010). B. Location of DYRK1A-specific guiding RNA sequences relative to genomic sequences of human and mouse DYRK1A. C. Human DYRK1A- KO U-2 OS clones. Sequencing analysis of the DYRK1A gene region targeted by CRISPR/Cas9 mutagenesis confirms presence of an extended insertion/deletion lesion in the KO clone #1, and a single nucleotide deletion in the KO clone #2. Genomic DNA region of interest was amplified by PCR, sequenced and aligned to genomic DYRK1A reference sequence using SnapGene software. WB analysis using two different DYRK1A antibodies confirms the loss of the full-length protein. D. Same as in panel C, only with mouse Dyrk1a-KO NIH3T3 clones. Figure S6. Characterization of the DYRK1A-KO U-2 OS cells. A. WB analysis of the indicated DNA damage signaling factors in the control (C) and DYRK1A-KO cells (clone #1) at different times after γ-irradiation (5 Gy). Vinculin and β-actin serve as loading controls for high and low molecular weight proteins, respectively. Note elevated expression of 53BP1 and BRCA1 in DYRK1A-KO cells both before and after irradiation. B. Propidium iodide (PI) stained DNA FACS analysis of the control and DYRK1A-KO U-2 OS cells reveals similar cell cycle changes in response to DNA damage. Figure S7. Characterization of the DYRK1A-KO U-2 OS cells (continued). A. DYRK1A-KO U-2 OS cells (clone #1) display normal patterns of the γH2AX and ubiquitylated protein accumulation at the sites of DNA damage. Cells were processed for staining with anti-γH2AX and anti-ubiquitylated protein antibodies (FK2) at 3h post-γ-irradiation (5 Gy). B. Recruitment of BRCA1 to the DSB sites appears normal in DYRK1A-KO U-2 OS cells (clone #1). The cells were treated and processed as in panel A. Figure S8. A. WB detection of adeno-SceI (HA-tagged) expression in the control and DYRK1A-KO U-2 OS cells. DYRK1A and vinculin are shown as controls. B. Representative FACS data show higher fraction of the GFP-positive cells in Ad-SceI-infected DYRK1A-KO U-2 OS cells compared to controls. Supplemental References Glenewinkel, F., Cohen, M.J., King, C.R., Kaspar, S., Bamberg-Lemper, S., Mymryk, J.S., and Becker, W. (2016). The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2. Scientific reports 6, 28241. Pierce, A.J., Johnson, R.D., Thompson, L.H., and Jasin, M. (1999). XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13, 2633-2638. Poulsen, M., Lukas, C., Lukas, J., Bekker-Jensen, S., and Mailand, N. (2012). Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks. J Cell Biol 197, 189-199. Varjosalo, M., Keskitalo, S., Van Drogen, A., Nurkkala, H., Vichalkovski, A., Aebersold, R., and Gstaiger, M. (2013). The protein interaction landscape of the human CMGC kinase group. Cell reports 3, 1306-1320. Yabut, O., Domogauer, J., and D'Arcangelo, G. (2010). Dyrk1A overexpression inhibits proliferation and induces premature neuronal differentiation of neural progenitor cells. J Neurosci 30, 4004-4014. Yakovlev, V.A. (2013). Nitric oxide-dependent downregulation of BRCA1 expression promotes genetic instability. Cancer Res 73, 706-715. Zheng, H., Ying, H., Wiedemeyer, R., Yan, H., Quayle, S.N., Ivanova, E.V., Paik, J.H., Zhang, H., Xiao, Y., Perry, S.R., et al. (2010). PLAGL2 regulates Wnt signaling to impede differentiation in neural stem cells and gliomas. Cancer Cell 17, 497-509. Menon et al., Supplemental Figure 1 (related to Fig. 1) Varjosalo only Overlap A (10) (14) ABCD3 DCAF7* CCDC8 DYNLL2 CCNA2 FAM117A CREBBP FAM117B DYNLL1 FAM53C EP300* FNTA FNTB GLCCI1 RB1* KIAA0232 RBL1* LZTS2 RBL2* PPP1R3F PRKAR1A RNF169 SIPA1L1 TROAP B IP: IP: IP: IP: Input DYRK1A Input DYRK1A Input DYRK1A Input DYRK1A FAM117B-CTAP FAM117B-CTAP FAM117B-CTAP DCAF7-CTAP DCAF7-CTAP DCAF7-CTAP GFP-CTAP GFP-CTAP GFP-CTAP GFP-CTAP GFP-CTAP GFP-CTAP LZTS2-CTAP LZTS2-CTAP LZTS2-CTAP GFP-CTAP GFP-CTAP GFP-CTAP NTAP-TROAP NTAP-TROAP NTAP-TROAP GFP-CTAP GFP-CTAP GFP-CTAP HA DYRK1A C IP: FAM117B FAM117B IP: Input IP: IgG Input IP: IgG IP: LZTS2 Input TROAP IP: IP: IgG IP: IgG Input IP: IgG IP: IgG IP: DYRK1A DYRK1A IP: DYRK1A DYRK1A DYRK1A DYRK1A DCAF7 FAM117B LZTS2 TROAP Menon et al., Supplemental Figure 2 (related to Fig. 2) Vector DYRK1A-WT DYRK1A-KR 53BP1 DAPI 53BP1+ Menon et al., Supplemental Figure 3 (related to Fig. 5) A WT S368D/S403D (DD) HA-RNF169 B C 90 n.s. 100 14 75 12 n.s. 80 10 60 * 60 8 45 6 40 30 4 HA Foci/Nucleus HA 15 20 2 Foci % Cells>10 HA % Cells>10 53BP1 Foci 0 0 0 WT DD WT DD Mock WT DD D E IP:HA Input IP: HA UIR 15 Gy, 3h UIR 15 Gy, 3h HA-RNF169 - WT AA DD Input K63 HA-RNF169 - WT AA DD - WT AA DD - WT AA DD - WT AA DD Ubiquitin USP7 HA β-actin HA HA (Input) Menon et al., Supplemental Figure 4 Related to Fig. 5) A DYRK1A HA-RNF169 β-actin 25 75 * 60 20 * 45 15 30 10 5 RNF169 Foci 15 %Cells with >10 HA- 0 0 HA-RNF169 Foci/Nucleus B DYRK1A β-actin 100 25 * 20 75 * 15 50 10 25 5 0 53BP1 Foci/Nucleus 0 Cells with >10 53BP1 Foci Menon et al., Supplemental Figure 5 (related to Fig. 6) A B RP11- 98O13 ! RP11-61A21 ! Genomic DNA! gRNA binding! RP11- 24M21 ! RP11- 105O24 ! hDYRK1A gRNA! 37486469 – RP11- 98O13 ! Homo sapiens chromosome 21, 37486488" RP11-61A21 ! GRCh38.p2 Primary Assembly; " RP11- 24M21 ! RP11- 105O24 ! DYRK1A; NC_000021.9 " " " mDYRK1A gRNA! 94665960 –" Mus musculus strain C57BL/6J 94665979" chromosome 16, GRCm38.p4 C57BL/ " 6J; DYRK1A; NC_000082.6" " " " CST D30C10 Bethyl 303-801A (around N523) (N-terminus) kDa KO #1 KO #2 Parental Control KO #1 #2 KO Parental Control C 100 DCAF7 Kinase HIS DYRK1A 75 93-104 159-479 599-619 50 37 25 1 NLS 763 20 117-134 15 10 Actin KO #2 KO #1 +112 bp del8 bp Sigma 7D10 CST D30C10 (C-Terminus) (around N523) KO #1 #2 KO Control KO #1 KO KO #2 kDa Control 100 DCAF7 Kinase HIS DYRK1A 75 93-104 159-479 599-619 D 50 37 1 NLS 763 25 117-134 20 15 Actin KO #1 KO #2 Menon et al., Supplemental Figure 6 (related to Fig.
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