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• W2077676818 abstract "Infection with human T-cell leukemia virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1, and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation-induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization, and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DNA damage response factor recruitment repressed the formation of ionizing radiation-induced Nbs1-containing foci. The Tax-induced “pseudo” DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for virus-induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response. Infection with human T-cell leukemia virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1, and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation-induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization, and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DNA damage response factor recruitment repressed the formation of ionizing radiation-induced Nbs1-containing foci. The Tax-induced “pseudo” DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for virus-induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response. Viruses have developed many host interaction strategies to acquire control of their environment. Some virus-host relationships progress through oncogenesis and result in cellular transformation. Among the so-called “transforming retroviruses,” the human T-cell leukemia virus, type I (HTLV-1) 2The abbreviations used are: HTLV-1human T-cell leukemia virus, type IDDRDNA damage responseIRionizing radiationIRIFIR-induced fociMRNMre11-Rad50-Nbs1TSSTax speckled structure(s)Gygraymono-ub-γ-H2AXmonoubiquitylation of γ-H2AX. displays a unique path through oncogenesis. HTLV-1 is the causative agent for adult T-cell leukemia, an aggressive leukemia/lymphoma. Infection by HTLV-1 is characterized by a long asymptomatic period of on average ∼30 years (1Proietti F.A. Carneiro-Proietti A.B. Catalan-Soares B.C. Murphy E.L. Oncogene. 2005; 24: 6058-6068Crossref PubMed Scopus (704) Google Scholar). Over this period of time ∼3% will develop adult T-cell leukemia (2Tajima K. Jpn. J. Cancer Res. 1998; 89 (inside front cover)Crossref Scopus (33) Google Scholar). The viral transactivator protein Tax has been shown to transform fibroblasts (3Pozzatti R. Vogel J. Jay G. Mol. Cell Biol. 1990; 10: 413-417Crossref PubMed Scopus (198) Google Scholar, 4Tanaka A. Takahashi C. Yamaoka S. Nosaka T. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 1071-1075Crossref PubMed Scopus (368) Google Scholar) and T-cell isolates (5Akagi T. Shimotohno K. J. Virol. 1993; 67: 1211-1217Crossref PubMed Google Scholar) and to spontaneously immortalize primary T-cells (6Grassmann R. Berchtold S. Radant I. Alt M. Fleckenstein B. Sodroski J.G. Haseltine W.A. Ramstedt U. J. Virol. 1992; 66: 4570-4575Crossref PubMed Google Scholar). The molecular details of the transformation process are not completely understood, but mounting evidence supports the hypothesis that Tax induces genomic instability in the host, a condition that accelerates accumulation of mutations that support cellular transformation (7Jeang K.T. Giam C.Z. Majone F. Aboud M. J. Biol. Chem. 2004; 279: 31991-31994Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 8Marriott S.J. Semmes O.J. Oncogene. 2005; 24: 5986-5995Crossref PubMed Scopus (146) Google Scholar). In fact, Tax expression directly correlates with the presence of genomic instability in pretransformed infected T-cells examined ex vivo (9Sibon D. Gabet A.S. Zandecki M. Pinatel C. Thête J. Delfau-Larue M.H. Rabaaoui S. Gessain A. Gout O. Jacobson S. Mortreux F. Wattel E. J. Clin. Invest. 2006; 116: 974-983Crossref PubMed Scopus (52) Google Scholar). human T-cell leukemia virus, type I DNA damage response ionizing radiation IR-induced foci Mre11-Rad50-Nbs1 Tax speckled structure(s) gray monoubiquitylation of γ-H2AX. We and others have proposed that Tax disrupts genomic integrity by repressing aspects of the cellular DNA damage response (DDR). Support for this hypothesis derives primarily from studies demonstrating that Tax expression results in increased cellular mutational frequency (10Kamada N. Sakurai M. Miyamoto K. Sanada I. Sadamori N. Fukuhara S. Abe S. Shiraishi Y. Abe T. Kaneko Y. Shimoyama M. Cancer Res. 1992; 52: 1481-1493PubMed Google Scholar, 11Miyake H. Suzuki T. Hirai H. Yoshida M. Virology. 1999; 253: 155-161Crossref PubMed Scopus (76) Google Scholar), inhibition of DNA repair (12Jeang K.T. Widen S.G. Semmes 4th, O.J. Wilson S.H. Science. 1990; 247: 1082-1084Crossref PubMed Scopus (297) Google Scholar, 13Kao S.Y. Marriott S.J. J. Virol. 1999; 73: 4299-4304Crossref PubMed Google Scholar, 14Philpott S.M. Buehring G.C. J. Natl. Cancer Inst. 1999; 91: 933-942Crossref PubMed Scopus (106) Google Scholar), and uncoupling of checkpoint control (15Haoudi A. Daniels R.C. Wong E. Kupfer G. Semmes O.J. J. Biol. Chem. 2003; 278: 37736-37744Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 16Haoudi A. Semmes O.J. Virology. 2003; 305: 229-239Crossref PubMed Scopus (42) Google Scholar, 17Kasai T. Iwanaga Y. Iha H. Jeang K.T. J. Biol. Chem. 2002; 277: 5187-5193Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Park H.U. Jeong J.H. Chung J.H. Brady J.N. Oncogene. 2004; 23: 4966-4974Crossref PubMed Scopus (57) Google Scholar). Sustained deficiencies of this sort are expected to result in genomic instability, and this hypothesis is consistent with the observation that genomic instability characterizes pretransformed HTLV-infected cells and the long asymptomatic period prior to transformation. An important cellular strategy for coordination of an efficient DDR is through the sequential recruitment of repair-response factors to the sites of DNA damage. The formation of these damage foci result from cellular efforts to consolidate repair activity and as such the formation, enumeration/size, and persistence of the foci reflect regulation of DDR (19Jackson S.P. Bartek J. Nature. 2009; 461: 1071-1078Crossref PubMed Scopus (3610) Google Scholar, 20Lukas C. Bartek J. Lukas J. Chromosoma. 2005; 114: 146-154Crossref PubMed Scopus (91) Google Scholar, 21Misteli T. Soutoglou E. Nat. Rev. Mol. Cell Biol. 2009; 10: 243-254Crossref PubMed Scopus (353) Google Scholar). The best studied of these nuclear structures is ionizing radiation-induced foci (IRIF). Immediately following insult by ionizing radiation, damaged DNA ends are recognized by the Mre11-Rad50-Nbs1 (MRN) complex. This recognition step is critical to the initial activation of DNA-PK/ATM, which sets in motion the activation of a number of downstream signal mediators such as Chk2, BRCA1, and 53BP1. The signaling coincides with recruitment of factors to the sites of damage, a process that is iterative and tied to the extent of damage. Recently, the recruitment of MDC1 (mediator of DNA damage checkpoint protein 1) to IRIF has been revealed as a critical event for initiation, amplification, and stabilization of the foci at sites of damage (22Dimitrova N. de Lange T. Genes Dev. 2006; 20: 3238-3243Crossref PubMed Scopus (75) Google Scholar, 23Eliezer Y. Argaman L. Rhie A. Doherty A.J. Goldberg M. J. Biol. Chem. 2009; 284: 426-435Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 24Lou Z. Minter-Dykhouse K. Franco S. Gostissa M. Rivera M.A. Celeste A. Manis J.P. van Deursen J. Nussenzweig A. Paull T.T. Alt F.W. Chen J. Mol. Cell. 2006; 21: 187-200Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 25Minter-Dykhouse K. Ward I. Huen M.S. Chen J. Lou Z. J. Cell Biol. 2008; 181: 727-735Crossref PubMed Scopus (69) Google Scholar, 26Spycher C. Miller E.S. Townsend K. Pavic L. Morrice N.A. Janscak P. Stewart G.S. Stucki M. J. Cell Biol. 2008; 181: 227-240Crossref PubMed Scopus (183) Google Scholar, 27Stucki M. Jackson S.P. DNA Repair. 2006; 5: 534-543Crossref PubMed Scopus (321) Google Scholar, 28Xie A. Hartlerode A. Stucki M. Odate S. Puget N. Kwok A. Nagaraju G. Yan C. Alt F.W. Chen J. Jackson S.P. Scully R. Mol. Cell. 2007; 28: 1045-1057Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The mobilization and coordination of IRIF formation is achieved in large part by ubiquitin-mediated events following the recruitment of the E3 ligase RNF8 (ring finger protein 8) by MDC1 to the foci (29Huen M.S. Grant R. Manke I. Minn K. Yu X. Yaffe M.B. Chen J. Cell. 2007; 131: 901-914Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar, 30Kolas N.K. Chapman J.R. Nakada S. Ylanko J. Chahwan R. Sweeney F.D. Panier S. Mendez M. Wildenhain J. Thomson T.M. Pelletier L. Jackson S.P. Durocher D. Science. 2007; 318: 1637-1640Crossref PubMed Scopus (694) Google Scholar, 31Marteijn J.A. Bekker-Jensen S. Mailand N. Lans H. Schwertman P. Gourdin A.M. Dantuma N.P. Lukas J. Vermeulen W. J. Cell Biol. 2009; 186: 835-847Crossref PubMed Scopus (151) Google Scholar, 32Yan J. Jetten A.M. Cancer Lett. 2008; 271: 179-190Crossref PubMed Scopus (65) Google Scholar). In the present study we demonstrate that Tax tethers MDC1 to chromatin overlapping with nuclear foci called Tax speckled structures (TSS). The TSS foci and IRIF do not colocalize, and recruitment of MDC1 is competitive between the two sites. We show that TSS contain the DDR proteins DNA-PK, BRCA1, and MDC1 and that Tax expression alone generates a DDR signal that is dependent upon the sequestration of MDC1. Tax expression and formation of TSS foci impaired the normal DDR as demonstrated through a quantitative reduction in damage-induced Nbs1-containing foci. These results support a novel competition model for Tax-induced genomic instability. 293T cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (Cambrex, East Rutherford, NJ) and 1% penicillin-streptomycin (Invitrogen). Transfections of 293T cells were performed by standard calcium phosphate precipitation. Generation of the STaxGFP, STax, and SGFP plasmids has been described (33Durkin S.S. Ward M.D. Fryrear K.A. Semmes O.J. J. Biol. Chem. 2006; 281: 31705-31712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). HA-tagged human MDC1 (pcDNA3-neo) was a gift from D. Lukas (Danish Cancer Society, Copenhagen, Denmark). For immunofluorescence analysis we used the following antibodies: anti-MDC1 goat polyclonal (1/100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-BrdU mouse monoclonal (1/50; BD Biosciences, San Jose, CA), anti-BRCA1 mouse monoclonal (1/100; Santa Cruz Biotechnology), anti-Nbs1 rabbit polyclonal (1/400; Novus Biologicals, Littleton, CO), and anti-DNA-PK Thr(P)2609 mouse monoclonal (1/400; Abcam, Cambridge, MA). For Western analysis, the following antibodies were used: anti-MDC1 goat polyclonal (1/200; Santa Cruz Biotechnology), anti-GFP mouse monoclonal (1/500; Santa Cruz Biotechnology), anti-HA rabbit polyclonal (1/1500, Zymed Laboratories Inc., San Francisco, CA), anti-tubulin mouse monoclonal (1/2000; Sigma), anti-Tax-pep3 rabbit polyclonal (1/2000), anti-ORC2 rabbit polyclonal (1/400; Santa Cruz Biotechnology), and anti-γ-H2AX (1/2000; R & D Systems, Minneapolis, MN). The cells were plated at 2 × 105 cells/ml in 60-mm plates. The following day the medium was replaced with serum-free and antibiotic-free medium 1 h prior to transfection. A total of 100 pmol of siRNA MDC1 (Santa Cruz Biotechnology) or siRNA control were diluted in 500 μl of Opti-MEM® I medium without serum. LipofectaminTM 2000 was gently mixed, and 5 μl was diluted in 500 μl of Opti-MEM® I medium without serum. The mixture was mixed and incubated for 5 min at room temperature. The selected DNA and/or siRNA were mixed with the diluted LipofectaminTM 2000 and incubated for 20 min at room temperature. The DNA-siRNA-LipofectaminTM 2000 complexes were then added to the plate. The plates were then incubated at 37 °C for 6 h, washed, and plated with complete medium. The cells were harvested after an additional 48 h. The cells were transfected directly upon coverslips. The medium was removed, and the coverslips were washed twice with ice-cold PBS. The cells were fixed with 4% paraformaldehyde for 12 min at room temperature. The fixed cells were then washed three times with PBS and incubated with ice-cold methanol for 2 min. The coverslips were then washed four times with PBS and incubated with primary antibody diluted in 3% BSA in PBS. Excess primary antibody was removed with two washes each of PBS, 1% Tween 20 and PBS. The coverslips were then incubated with species-specific Alexa Fluor 594-conjugated secondary antibody (Invitrogen) diluted at 1/1000 in 3% BSA in PBS. The nuclei were counterstained with To-Pro-3 iodide (Invitrogen) diluted 1/1,000 in the same secondary antibody solution. Excess secondary antibody was removed with two washes each of 3% BSA in PBS and PBS. One drop of Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA) was placed onto a slide, and the coverslip was inverted onto the slide and left to air dry for 1 h at room temperature in the dark. Confocal fluorescent images were acquired using a Zeiss LSM 510 confocal microscope at 63× magnification with a 2× zoom using argon (488 nm), HeNe1 (543 nm), and HeNe2 (633 nm) lasers and imaged with LSM Image Browser software (Carl Zeiss, Jena, Germany). Proteins separated by SDS-PAGE were transferred to Immobilon-P membranes (Millipore, Billerica, MA) using the semi-dry transfer method with 20 V applied for 1 h. The membranes were then blocked for 1 h at room temperature in 1× Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE). Primary antibodies diluted in 1× Odyssey blocking buffer were applied to the membranes and incubated at 4 °C overnight with shaking. The membranes were washed four times for 5 min with PBS, 1% Tween 20. Li-Cor Odyssey secondary antibodies were diluted to a concentration of 1/20,000 in 1× Odyssey blocking buffer containing 0.5% SDS and 0.5% Tween and then incubated for 1 h at room temperature protected from light. The membranes were washed four times for 5 min with PBS, 1% Tween 20 and then stored in PBS until ready to be analyzed. The blots were scanned and analyzed with a Li-Cor Odyssey scanner and software. Approximately 5 × 106 cells were rinsed twice with ice-cold PBS and harvested with a cell scraper into 1 ml of ice-cold PBS. The cells were centrifuged at 1500 rpm for 2 min, the supernatant was discarded, and the cell pellet was resuspended in ice-cold PBS. The cells were again centrifuged, and the pellet was resuspended in 200 μl of buffer A (100 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.34 m sucrose, 10% glycerol, 10 mm NaF, 1 mm Na2VO3, 1 mm DTT, and protease inhibitor mixture) with a blunt 1000-μl micropipette tip. Triton X-100 was added to a final concentration of 0.1%, and the solution was incubated on ice for 5 min. The solution was then centrifuged at 1,300 × g (4000 rpm) at 4 °C for 5 min in a 1.5-ml centrifuge tube. The supernatant (fraction S1) was separated from the pellet (fraction P1), which contains the nuclei. Fraction S1 was clarified by high speed centrifugation at 20,000 × g (14,000 rpm) at 4 °C for 5 min. The supernatant (fraction S2), which represents the cytosolic fraction, was collected, and the pellet was discarded. Fraction S2 was stored at −80 °C until ready to use. Pellet fraction P1 was washed once with buffer A (0.6 ml/tube) by centrifugation again at 4000 rpm at 4 °C for 5 min. Washed fraction P1 was resuspended in 100 μl of buffer B (3 mm EDTA, 0.2 mm EGTA, 1 mm DTT, protease inhibitor mixture) with a blunt 1000-μl micropipette tip and lysed for 30 min on ice. The P1 sample was then centrifuged at 1,700 × g (5000 rpm) at 4 °C for 5 min. The resulting supernatant (fraction S3), which contains the soluble nuclear proteins, was separated from the pellet (fraction P2) that contains the chromatin. Fraction S3 was stored at −80 °C until ready to use. Fraction P2 was washed once with Buffer B (0.6 ml/tube) by centrifugation at 10,000 × g (11,000 rpm) at 4 °C for 1 min. Fraction P2 was then resuspended in M-PER (Thermo-Fisher Scientific, Rockford, IL) protein extraction buffer (180 μl/sample), briefly sonicated, and clarified by high speed centrifugation at 20,000 × g (14,000 rpm) at 4 °C for 5 min. The final supernatant (fraction P3), which contains the chromatin-bound proteins, was collected and kept at −80 °C until ready to use. Mock and STaxGFP transfected cells were incubated with 10 μg/ml of BrdU (Sigma) for 30 h under normal cell culture conditions and then exposed to 15 Gy of x-ray irradiation or left untreated. After ionizing radiation, the cells were grown for 16 h, washed with PBS, fixed with methanol for 5 min at −20 °C, and blocked with 3% BSA for 30 min. The cells were immunostained with mouse anti-BrdU followed by anti-mouse Alexa Fluor 594-conjugated secondary antibody (Invitrogen). The nuclei were counterstained with To-Pro-3 iodide (Invitrogen) diluted at 1/1,000. We previously described the subcellular localization of Tax to prominent nuclear speckles we termed TSS (34Semmes O.J. Jeang K.T. J. Virol. 1996; 70: 6347-6357Crossref PubMed Google Scholar) and have subsequently demonstrated the recruitment of Chk2 and DNA-PK to TSS by Tax (15Haoudi A. Daniels R.C. Wong E. Kupfer G. Semmes O.J. J. Biol. Chem. 2003; 278: 37736-37744Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 35Durkin S.S. Guo X. Fryrear K.A. Mihaylova V.T. Gupta S.K. Belgnaoui S.M. Haoudi A. Kupfer G.M. Semmes O.J. J. Biol. Chem. 2008; 283: 36311-36320Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Gupta S.K. Guo X. Durkin S.S. Fryrear K.F. Ward M.D. Semmes O.J. J. Biol. Chem. 2007; 282: 29431-29440Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These observations and the knowledge that Tax expression results in genomic instability led us to speculate that Tax induces genomic instability via functional sequestration of proteins of the cellular DDR. To advance this hypothesis, we examined additional DDR proteins for colocalization to TSS. Tax-GFP fusion protein was transiently expressed, and endogenous DDR proteins were analyzed for colocalization using indirect immunofluorescence confocal microscopy. In Fig. 1, we show a typical expression pattern of Tax localized to nuclear TSS. The phosphatidylinositol 3-kinase damage response activator DNA-PK, activated via phosphorylation at Thr2609, colocalizes with TSS as we have previously demonstrated (33Durkin S.S. Ward M.D. Fryrear K.A. Semmes O.J. J. Biol. Chem. 2006; 281: 31705-31712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In addition, we now show that the DDR mediator proteins BRCA1 and MDC1 also localize to TSS. Greater than 80% of all nuclei counted showed colocalization between TSS and the indicated protein. Foci for BRCA1, DNA-PK, and MDC1 were observed in the absence of Tax expression in less than 5% of cells. The localization of nucleolin to a nuclear domain distinct from TSS is included as a control. The recruitment of MDC1 to TSS is especially interesting given the critical role this protein plays in the initiation and amplification of DDR, and IRIF formation and maintenance. These findings establish that Tax-induced TSS foci are enriched for DDR proteins. To gain insight into the formation of TSS, it was critical to determine whether the TSS were independent/separable from classic DDR structures such as IRIF. Because TSS share many of the protein markers of IRIF, we utilized a modified BrdU staining technique that detects DNA breaks to visualize damage foci (37Raderschall E. Golub E.I. Haaf T. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1921-1926Crossref PubMed Scopus (286) Google Scholar). Tax-expressing cells were pulsed with BrdU for 30 h before ionizing radiation treatment to completely label the DNA. No denaturation step was used before anti-BrdU staining; therefore only the accessible BrdU epitopes that occur at DNA strand breaks are visualized. If TSS are formed at sites of DNA damage, then we would expect to observe colocalization with the BrdU foci. As shown in Fig. 2, in the absence of exogenous irradiation, there were no nuclear BrdU foci and thus no evidence of TSS residing at sites of DNA damage. We next induced the formation of IRIF by exposing the cells to 5 Gy of IR and examined the cells 16 h later. In the Tax-expressing cells exposed to IR clear BrdU damage, foci were observed. However, there was only incidental overlap between TSS and IRIF. Specifically, less than 50% of cells expressing both foci displayed overlap and in those cells that did, ∼7% of TSS contained BrdU foci. These data demonstrate that although TSS and IRIF share many DDR proteins, they are in fact different structures. Having established that TSS and IRIF are independent structures, we hypothesized that TSS may compete with IRIF for limiting DDR proteins. This competition for limiting DDR proteins would provide a mechanism for repression of the repair response and Tax-mediated genomic instability. One approach toward identification of limiting factors is to induce both TSS and IRIF and observe the distribution of known DDR proteins. In these studies, we expressed Tax-GFP and examined the colocalization of endogenous proteins by indirect immunofluorescence confocal microscopy. We established IRIF by exposure to 5 Gy of IR to ensure maximum recruitment to IRIF. In Fig. 3 we show that MDC1, DNA-PK, and BRCA1 are all induced to localize to TSS in the absence of IR. Greater than 80% of counted cells showed colocalization of TSS and each of these nuclear foci. When both TSS and IRIF are present, DNA-PK and BRCA1 are effectively recruited to both structures in >92% of cells. However, when presented with both IRIF and TSS, MDC1 localized almost exclusively to foci outside of TSS. This result suggests that MDC1 may be a critical factor for regulating TSS/IRIF formation. Interestingly, expression of Tax did not result in induction of Nbs1 localization to TSS in the absence of IR, although we could demonstrate efficient induction and localization to IRIF. Because Nbs1 is a critical component of the damage-sensing complex MRN, the absence of Nbs1 in TSS is consistent with a lack of associated DNA breaks. Of the tested DDR proteins, MDC1 appears to localize either to TSS in the absence of IR or to IRIF in the presence of high doses of IR. We reasoned that if the localization of MDC1 to TSS and/or IRIF was competitive, then MDC1 localization would be dictated by the relative recruitment to each site. We were unable to effectively titrate IRIF directly because IR exposures less than 1 Gy resulted in inconsistent formation of IR-induced MDC1-containing foci. Therefore, to achieve a competition balance between the amount of damage versus the amount of Tax, we “titrated” damaged DNA by following time points post-repair. We reasoned that as damage was resolved then the “demand” for IR foci would fall below the demand for Tax foci. A standard dose of 1 Gy was found to produce sufficient IRIF and allow for cell recovery. Because the experiments were conducted within 24 h, Tax expression was relatively constant (protein half-life, >24 h). Each experimental time point resulted from analysis of 25 nuclei from cells in which both TSS and IRIF were observed. A simple numerical average was generated for the percentage of foci that overlap. In the absence of IR the colocalization of MDC1 to TSS was >84% (Fig. 4). However, at 4 h following exposure to 1 Gy of IR, MDC1 dramatically relocalized to IRIF with <9% of foci overlap. We also observed that the relative size and shape of the TSS remain unchanged throughout the experiment. At the 24-h time point the MDC1 foci outside of TSS have both reduced in number and size as is consistent with resolved DNA damage. Under these conditions, ∼47% of the TSS now displayed colocalization with MDC1. Thus, as damaged DNA is repaired and the recruitment balance shifts toward TSS foci, MDC1 relocalizes to TSS. Damage-independent DDR foci were shown to result from tethering MDC1 to DNA (38Soutoglou E. Misteli T. Science. 2008; 320: 1507-1510Crossref PubMed Scopus (246) Google Scholar). We thus speculated that Tax initiates a damage-independent DRR within TSS by binding to and recruiting MDC1. In this series of experiments we sought to establish whether Tax binds to MDC1. HA-MDC1 and either S-TaxGFP, S-Tax, or S-GFP were cotransfected into 293T cells, and Tax complexes were subsequently isolated from whole cell lysates using S-agarose chromatography. We directly normalized cell lysates to expression of MDC1 and adjusted expression efficiency to achieve comparable expression of Tax, Tax-GFP, or GFP. We also conducted immunoblot analysis for expression of tubulin in the extracts prior to purification of complexes for additional experimental normalization. In Fig. 5A, we show that both TaxGFP and Tax protein coprecipitates with MDC1 at comparable efficiency. In contrast, GFP fails to form a complex with MDC1. These results demonstrate a specific binding between Tax and MDC1. We previously determined that TSS reside within the subnuclear architecture of interchromatin granules (34Semmes O.J. Jeang K.T. J. Virol. 1996; 70: 6347-6357Crossref PubMed Google Scholar). Thus, if Tax is binding to MDC1 and recruiting this protein into TSS, then the relative amount of MDC1 within the chromatin fraction should increase in the presence of Tax. In this experiment, we isolated the cellular chromatin fraction from cells either expressing Tax or not. The isolated chromatin fractions were then separated by SDS-PAGE and immunoblotted to determine steady-state levels of endogenous MDC1. We initially determined that the total expression of MDC1 was unaffected by the expression of Tax (Fig. 5B). When normalized to tubulin, the total cell extracts presented with equivalent levels of MDC1. However, in the chromatin fraction the relative amount of MDC1 was dramatically increased in the Tax-expressing cells (Fig. 5C). As has been reported elsewhere, we see multiple forms of MDC1 with different migration rates (39Stucki M. Clapperton J.A. Mohammad D. Yaffe M.B. Smerdon S.J. Jackson S.P. Cell. 2005; 123: 1213-1226Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar). It has been speculated that these isoforms are due to differential phosphorylation (40Lou Z. Minter-Dykhouse K. Wu X. Chen J. Nature. 2003; 421: 957-961Crossref PubMed Scopus (287) Google Scholar). The chromatin extracts were normalized to expression of the chromatin-specific protein Orc2. The relative expression of tubulin and Orc2 was used to qualify the efficiency of the chromatin fractionation process. Thus, Tax-expressing cells display an increased steady-state level of endogenous MDC1 in the chromatin fraction as a result of Tax expression. This result is consistent with our model for Tax binding to MDC1 and recruiting the protein to chromatin-associated TSS. We recently identified a TSS localization signal in Tax that is necessary and sufficient for targeting to TSS (41Fryrear K.A. Durkin S.S. Gupta S.K. Tiedebohl J.B. Semmes O.J. J. Virol. 2009; 83: 5339-5352Crossref PubMed Scopus (16) Google Scholar). Specifically, when we linked GFP to the first 75 amino acids of Tax, encompassing both the NLS (1–50) and TSS localization signal (50–75), this protein formed nuclear foci and targeted to the same subnuclear sites as wild type Tax. Having identified the minimal structure of Tax required for localization to TSS, we asked whether the TSS localization signal domain was able to recruit cellular DDR. We observed that although Tax1–75 could efficiently form TSS foci, neither MDC1 nor DNA-PK Thr(P)2609 was recruited to these structures (Fig. 6). We then examined the Tax 1–75 expressing cells for ability to form IRIF. The formation of IRIF and recruitment of MDC1 and DNA-PK to foci was unimpaired in these cells. In addition, the IRIF did not colocalize with the TSS. These data show that recruitment of DDR proteins to TSS requires the C-terminal half of Tax and suggests that Tax serves to bridge the formation of DDR-containing TSS. We reasoned that if TSS and IRIF compete for MDC1 recruitment and DDR activation, then expression of Tax should impair the normal damage-induced DDR. As we noted earlier, in contrast to all other accessed DDR factors, Nbs1 was not recruited to TSS foci. Therefore, we were able to measure IRIF formation separate from TSS formation by observation of Nbs1 recruitment into IRIF. In contrast to MDC1 foci, we were able to observe a robust Nbs1-containing foci formation at lower IR doses, and so for these experiments we utilized an exposure of 0.5 Gy. Exp" @default.
• W2077676818 created "2016-06-24" @default.
• W2077676818 creator A5001601904 @default.
• W2077676818 creator A5025982554 @default.
• W2077676818 creator A5058145167 @default.
• W2077676818 creator A5061424957 @default.
• W2077676818 creator A5085974923 @default.
• W2077676818 date "2010-10-01" @default.
• W2077676818 modified "2023-10-16" @default.
• W2077676818 title "The Viral Oncoprotein Tax Sequesters DNA Damage Response Factors by Tethering MDC1 to Chromatin" @default.
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