Role for KAP1 Serine 824 Phosphorylation and Sumoylation/Desumoylation Switch in Regulating KAP1-mediated Transcriptional Repression

As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein posttranslational modifications, including phosphorylation and sumoylation. However, gaps exist in our knowledge of how KAP1 phosphorylation cross-talks with KAP1 sumoylation and what the biological consequence is. Here, we show that doxorubicin (Dox) treatment induces KAP1 phosphorylation at Ser-824 via an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21WAF1/CIP1 and Gadd45α. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 sumoylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 sumoylation and stimulation of p21 transcription before the exposure of Dox. We further provide evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21 and Gadd45α transcription. Moreover, KAP1 and differential sumoylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its sumoylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses. As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein posttranslational modifications, including phosphorylation and sumoylation. However, gaps exist in our knowledge of how KAP1 phosphorylation cross-talks with KAP1 sumoylation and what the biological consequence is. Here, we show that doxorubicin (Dox) treatment induces KAP1 phosphorylation at Ser-824 via an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21WAF1/CIP1 and Gadd45α. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 sumoylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 sumoylation and stimulation of p21 transcription before the exposure of Dox. We further provide evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21 and Gadd45α transcription. Moreover, KAP1 and differential sumoylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its sumoylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses. The Krüppel-associated box zinc finger proteins (KRAB-ZFP) comprise approximately one-third of the 799 different zinc finger proteins, constituting the largest single-family transcriptional regulators in mammals (1Urrutia R. Genome Biology 2004. 2003; 4: 231Google Scholar). KRAB domain-associated protein 1 (KAP1) 3The abbreviations used are:KAP1KRAB domain-associated protein 1DoxdoxorubicinATMataxia telangiectasia mutatedATRAT Rad3-relatedEGFPenhanced green fluorescent proteinkdkinase-deadRTreverse transcriptionshshort hairpinSUMOsmall ubiquitin-like modifier. functions as transcriptional corepressor for ZBRK1, a KRAB-ZFP member, by acting as a transcription intermediary factor to connect KRAB-ZFPs to transcriptional repression machinery. Because KAP1 itself cannot bind DNA directly, the specificity of transcriptional repression is dictated by its interaction with ZBRK1 through protein-protein interaction. The RING finger-B box-coiled-coil domain of KAP1 associates with the KRAB domain of ZBRK1, repressing the transcription of DNA damage-responsive gene Gadd45α (2Zheng L. Pan H. Li S. Flesken-Nikitin A. Chen P.L. Boyer T.G. Lee W.H. Mol. Cell. 2000; 6: 757-768Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and p21WAF1/CIP1 (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). KAP1 can recruit and coordinate several components of gene silencing machinery. For example, KAP1 interacts with histone deacetylase complex NuRD and N-CoR1 and binds to histone methyltransferase SETDB1 to modify the configuration of chromatin structures (4Schultz D.C. Ayyanathan K. Negorev D. Maul G.G. Rauscher F.J. II I Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (914) Google Scholar, 5Schultz D.C. Friedman J.R. Rauscher F.J. II I Genes Dev. 2001; 15: 428-443Crossref PubMed Scopus (408) Google Scholar, 6Underhill C. Qutob M.S. Yee S.P. Torchia J. J. Biol. Chem. 2000; 275: 40463-40470Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). KAP1 also recruits heterochromatin protein 1 (HP1) to histones through a PXVXL motif (7Lechner M.S. Begg G.E. Speicher D.W. Rauscher F.J. II I Mol. Cell. Biol. 2000; 20: 6449-6465Crossref PubMed Scopus (169) Google Scholar, 8Sripathy S.P. Stevens J. Schultz D.C. Mol. Cell. Biol. 2006; 26: 8623-8638Crossref PubMed Scopus (233) Google Scholar). In addition, KAP1 is identified as a Mdm2-binding protein that inactivates p53 (9Okamoto K. Kitabayashi I. Taya Y. Biochem. Biophys. Res. Commun. 2006; 351: 216-222Crossref PubMed Scopus (61) Google Scholar, 10Wang C. Ivanov A. Chen L. Fredericks W.J. Seto E. Rauscher F.J. II I Chen J. EMBO J. 2005; 24: 3279-3290Crossref PubMed Scopus (192) Google Scholar). KRAB domain-associated protein 1 doxorubicin ataxia telangiectasia mutated AT Rad3-related enhanced green fluorescent protein kinase-dead reverse transcription short hairpin small ubiquitin-like modifier Emerging evidence supports the idea that post-translational modifications, including phosphorylation and sumoylation, play a pivotal role in regulating transcriptional control in response to different extracellular milieu. As a multifunctional protein, KAP1 is reportedly subjected to multiple post-translational modifications, and we have recently reported that sumoylation, a post-translational Lys modification, plays a major role in mediating KAP1 transcriptional co-repressor function and attenuating doxorubicin (Dox)-induced p21WAF1/CIP1 transcriptional activation in breast cancer MCF-7 cells (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). There are at least three Lys residues, 554, 779, and 804, that serve as the major sumoylation targets for KAP1, and the overall sumoylation capacity of KAP1 is transiently decreased upon Dox exposure. Moreover, the differential sumoylation status of KAP1 functions to modulate p21 transcription by switching the histone His-3–Lys-9 methylation and His-3–Lys-9 and -Lys-14 acetylation statuses without affecting the occupancy of the p21 proximal promoter by KAP1/ZBRK1 in MCF-7 cells. The KAP1 sumoylation-mimetic, SUMO-1-KAP1, desensitizes MCF-7 cells to Dox-induced cell death. Collectively, the KAP1 sumoylation/desumoylation switch suppresses KAP1 transcriptional co-repressor function by down-regulating histone His-3–Lys-9 methylation and fostering His-3–Lys-9 and -Lys-14 acetylation. However, it remains unclear what signal regulates the KAP1 sumoylation/desumoylation switch in response to Dox-induced DNA damage. A major response to DNA damage insults, such as DNA double-strand breaks by x-ray irradiation or Dox treatment, is the activation of the nuclear protein kinase ataxia telangiectasia mutated (ATM) (11Shiloh Y. Annu. Rev. Genet. 1997; 31: 635-662Crossref PubMed Scopus (428) Google Scholar, 12Shiloh Y. Trends Biochem. Sci. 2006; 31: 402-410Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Patients with the genome instability syndrome ataxia telangiectasia are very sensitive to ionization radiation due to the lack of proper DNA damage response (11Shiloh Y. Annu. Rev. Genet. 1997; 31: 635-662Crossref PubMed Scopus (428) Google Scholar). Upon the occurrence of double-strand breaks, ATM is rapidly phosphorylated, and ATM's own kinase activity subsequently phosphorylates a number of substrates, including H2AX, TopBP1, NBS1, and BRCA1 etc., resulting in the activation of cell cycle checkpoint control and DNA repair machinery (13Sancar A. Lindsey-Boltz L.A. Unsal-Kacmaz K. Linn S. Annu. Rev. Biochem. 2004; 73: 39-85Crossref PubMed Scopus (2557) Google Scholar). ATM belongs to a conserved protein family termed nuclear phosphatidylinositol 3-kinase protein-like kinases. Most members of the phosphatidylinositol 3-kinase protein-like kinase family possess serine/threonine kinase activity and a domain that is characteristic of phosphatidylinositol 3-kinase (14Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6315) Google Scholar). Besides ATM, ataxia telangiectasia and Rad3-related (ATR), hSMG-1, mTOR, and the catalytic subunit of DNA-PK are the four other protein kinases of this family identified so far. Numerous studies have established that the biological response to genotoxic stresses in mammalian cells is to trigger a complex network of transcriptional activation by the checkpoint signaling kinases ATM and ATR and their effector kinases Chk2 and Chk1, respectively (for review, see Ref. 15Kastan M.B. Bartek J. Nature. 2004; 432: 316-323Crossref PubMed Scopus (2228) Google Scholar). One of the key consequences is cell cycle arrest and apoptosis, mediated by the induction of proteins involved in cell cycle control (p21WAF1/CIP1 and Gadd45α) and proapoptosis (Bax, Puma, and Noxa) (16el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar, 17Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1302) Google Scholar, 18Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2738) Google Scholar). The balance of the combined induction of these genes leads to either cell cycle arrest or apoptosis when proapoptotic genes are transcribed at a level above threshold, depending on the severity of DNA damage. Very recently, O'Geen at al. (19O'Geen H. Squazzo S.L. Iyengar S. Blahnik K. Rinn J.L. Chang H.Y. Green R. Farnham P.J. PLoS Genet. 2007; 3: e89Crossref PubMed Scopus (150) Google Scholar) have identified ∼7000 KAP1 target sites by using chromatin immunoprecipitation assays coupled with a human 5-kilobase promoter array or a complete genomic tiling array. In light of the recent evidence for the multifunction of KAP1 (4Schultz D.C. Ayyanathan K. Negorev D. Maul G.G. Rauscher F.J. II I Genes Dev. 2002; 16: 919-932Crossref PubMed Scopus (914) Google Scholar, 5Schultz D.C. Friedman J.R. Rauscher F.J. II I Genes Dev. 2001; 15: 428-443Crossref PubMed Scopus (408) Google Scholar, 6Underhill C. Qutob M.S. Yee S.P. Torchia J. J. Biol. Chem. 2000; 275: 40463-40470Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 7Lechner M.S. Begg G.E. Speicher D.W. Rauscher F.J. II I Mol. Cell. Biol. 2000; 20: 6449-6465Crossref PubMed Scopus (169) Google Scholar, 8Sripathy S.P. Stevens J. Schultz D.C. Mol. Cell. Biol. 2006; 26: 8623-8638Crossref PubMed Scopus (233) Google Scholar, 9Okamoto K. Kitabayashi I. Taya Y. Biochem. Biophys. Res. Commun. 2006; 351: 216-222Crossref PubMed Scopus (61) Google Scholar, 10Wang C. Ivanov A. Chen L. Fredericks W.J. Seto E. Rauscher F.J. II I Chen J. EMBO J. 2005; 24: 3279-3290Crossref PubMed Scopus (192) Google Scholar, 19O'Geen H. Squazzo S.L. Iyengar S. Blahnik K. Rinn J.L. Chang H.Y. Green R. Farnham P.J. PLoS Genet. 2007; 3: e89Crossref PubMed Scopus (150) Google Scholar), it is tempting to speculate that the identified Dox-stimulated KAP1 desumoylation (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) could have a profound effect on the regulation of global gene expression in response to genotoxins. Moreover, KAP1 is demonstrated to be phosphorylated at Ser-824 by the phosphatidylinositol 3-kinase protein-like kinase family member of kinases after DNA damage insult (20White D.E. Negorev D. Peng H. Ivanov A.V. Maul G.G. Rauscher F.J. II I Cancer Res. 2006; 66: 11594-11599Crossref PubMed Scopus (107) Google Scholar, 21Ziv Y. Bielopolski D. Galanty Y. Lukas C. Taya Y. Schultz D.C. Lukas J. Bekker-Jensen S. Bartek J. Shiloh Y. Nat. Cell Biol. 2006; 8: 870-876Crossref PubMed Scopus (552) Google Scholar). Despite extensive literature investigating KAP1 function, little is known regarding the coordinated relieving of KAP1-mediated repression on p21, Gadd45α and other ZBRK1-binding element-containing genes by its multiple post-translational modifications, such as sumoylation and phosphorylation, in response to DNA damaging stimuli. Given that ATM regulates many aspects of DNA damage responses, including the induction of p21, we were intrigued by the possibility that ATM functions upstream of KAP1 SUMO-1 conjugation/deconjugation in response to DNA damage. Our results indicate that Dox treatment induces KAP1 Ser-824 phosphorylation and de-repression of Gadd45α and p21 transcription mainly via ATM, whereas the basal transcriptional co-repressor potential and sumoylation of KAP1 are coordinately regulated by SENP1 deSUMOylase. Finally, we demonstrate that the basal and DNA damage-induced transcription of a subset of KAP1-targeted genes, such as p21, Gadd45α, Bax, Puma, and Noxa, is coordinately regulated by the interplay of KAP1 sumoylation and phosphorylation. Our results could further unveil the role of a previously unnoticed ATM and KAP1 sumoylation switch in DNA damage responses. Cell Culture—HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin, and MCF-7 cells were cultured in the same medium with an addition of 0.01 mg/ml recombinant human insulin in a humidified atmosphere of 37 °C and 5% CO2. The stable KAP1 knockdown cell line K928-cI10 was grown as above with the addition of 10 μg/ml puromycin (8Sripathy S.P. Stevens J. Schultz D.C. Mol. Cell. Biol. 2006; 26: 8623-8638Crossref PubMed Scopus (233) Google Scholar). Both ATM-deficient pEBS7 and ATM-proficient YZ5 cells (22Ziv Y. Bar-Shira A. Pecker I. Russell P. Jorgensen T.J. Tsarfati I. Shiloh Y. Oncogene. 1997; 15: 159-167Crossref PubMed Scopus (223) Google Scholar) were maintained in Eagle's Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, antibiotics, 2 nm glutamine, 100 μg/ml hygromycin, and 1.25 units/ml nystatin in a humidified atmosphere of 37 °C and 5% CO2. GK41 cells, U2OS (human osteosarcoma) stably transfected with a doxycycline-inducible ATR-kd (kinase-dead) construct (23Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (263) Google Scholar), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus 50 units/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml hygromycin, and 200 μg/ml Geneticin. Western Analyses—Whole cell lysates were prepared by lysing cells with radioimmune precipitation assay buffer (25 mm Tris, 125 mm NaCl, 1% Nonidet-P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide, pH 8.0) plus Complete Protease Inhibitor Mixture (Roche Applied Science) containing 10 mm N-ethylmaleimide, 1 mm NaF, and 2 mm Na3VO4 and subjected to SDS-PAGE followed by immunoblotting with antibodies for FLAG-KAP1 (M2, Sigma-Aldrich), tubulin (D-10) (Santa Cruz Biotechnologies), phospho-Ser-824-KAP1 (A300–767A, Bethyl Laboratories), and EGFP (Santa Cruz Biotechnologies). Blots were visualized with an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Biosciences) and a Versadoc 5000 Imaging System (Bio-Rad). Densitometric data were obtained and analyzed with Quantity One Software (Bio-Rad). Results of Western analyses shown in this report are representative of two to four independent experiments. Luciferase Assays—The p21-Luc reporter construct was made by subcloning a 2.3-kilobase p21 promoter into pGL3-Basic as previously described (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The Gadd45α-Luc reporter was a gift from Dr. Wen-Hwa Lee at the University of California, Irvine. The luciferase reporters were co-transfected with a firefly control reporter, pRL-TK, for the purpose of normalization. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Luciferase assays were carried out with DualGlo Luciferase assay kit (Promega), and the desired luciferase activity was calculated after normalization against the co-transfected firefly luciferase activity. In Vivo Sumoylation Assays—The in vivo sumoylation assay was carried out with co-transfection of FLAG-KAP1 or its mutants and EGFP-SUMO-1 in a 1:4 ratio into HEK293 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. SUMOylated KAP1 was detected by immunoblotting of whole cell lysates against FLAG or EGFP tag. Immunoprecipitation of KAP1—Transfection of FLAG-KAP1 or FLAG-KAP1 with EGFP-SUMO-1 expression constructs into HEK293 or MCF-7 cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's manual. Whole cell lysates were prepared by lysing cells with lysis buffer (50 mm Tris-HCL, pH 6.8, 100 mm NaCl, 0.5 mm MgCl2, 1mm EDTA, 0.2% Nonidet P-40, 1 mm dithiothreitol, 1× protease inhibitor mixture (Roche Applied Science), 10 mm N-ethylmaleimide (Sigma-Aldrich). For each sample, 5 μl of anti-FLAG M2 antibody was mixed with 1 mg of whole cell lysates and incubated on ice for 2 h. Then Protein A/G PLUS-agarose (Santa Cruz Biotechnology, CA) was added, and the sample was rotated at 4 °C overnight. The mixture was then washed with 1 ml of 1× phosphate-buffered saline three times. Immunoprecipitates were then eluted in 40 μl of 2× SDS sample buffer, and half of the elution was subjected to immunoblotting analyses. Total RNA Extraction, Reverse Transcription, and Real-time PCR—Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The total RNA from Dox-treated and control MCF-7 cells was then treated with RNase-free DNase (Invitrogen) and extracted again with phenol-chloroform followed by ethanol precipitation. Reverse transcription and quantitative PCR of p21, Gadd45α, Bax, Puma, and Noxa mRNA were performed with iTaq SYBR Green Supermix (Bio-Rad), a fraction of each total RNA sample, and specific pairs of gene-specific primers (Table 1). PCR amplification and fluorescence detection were done with MyIQ real-time PCR detection system, and the threshold cycles were determined by iCycler program with its default setting. -Fold inductions were determined by the ΔΔCt method against 18 S rRNA.TABLE 1Primer pairs used in the real-time RT-PCR experimentsPrimerPurposeSequence (5′ to 3′)18 S rRNA FPReal-time PCRCGGCGACGACCCATTCGAAC18 S rRNA RPRT and real-time PCRGAATCGAACCCTGATTCCCCGTCp21 FPReal-time PCRTTTCTCTCGGCTCCCCATGTp21 RPRT and real-time PCRGCTGTATATTCAGCATTGTGGGGadd45α FPReal-time PCRAGGAAGTGCTCAGCAAAGCCGadd45α RPRT and real-time PCRGCACAACACCACGTTATCGGBax FPReal-time PCRCCGATTCATCTACCCTGCTGBax RPRT and real-time PCRCAATTCCAGAGGCAGTGGAGNoxa FPReal-time PCRATTACCGCTGGCCTACTGTGNoxa RPRT and real-time PCRGTGCTGAGTTGGCACTGAAAPuma FPReal-time PCRCTGTGAATCCTGTGCTCTGCPuma RPRT and real-time PCRAATGAATGCCAGTGGTCACA Open table in a new tab Constructs—Human SENP1 cDNA was amplified from testis cDNA library (Clontech) and cloned into EcoRI and EcoRV sites of pCMV-HA vector, yielding hemagglutinin-SENP1. Hemagglutinin (HA)-SENP1C603S mutant was constructed from HA-SENP1 plasmid by QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotides corresponding to SENP1 nucleotide sequence 413ACCATCACTGCCATGTATC431 and 520ACTCAGAGGCGACATGTTA538 were inserted into pSuper vector (Oligoengine) to generate sh-SENP1-1 and sh-SENP1–2, respectively. FLAG-KAP1(S824D), FLAG-KAP1(S824A), and SUMO-1-KAP1(L306P) mutants were engineered using FLAG-KAP1 and SUMO-1-KAP1 (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) as a template, respectively, by a QuikChange site-directed mutagenesis kit (Stratagene). Inducible sh-KAP1—Tet-inducible sh-KAP1 was constructed according to van de Wetering et al. (24van de Wetering M. Oving I. Muncan V. Pon Fong M.T. Brantjes H. van Leenen D. Holstege F.C. Brummelkamp T.R. Agami R. Clevers H. EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (463) Google Scholar). The oligonucleotides used were as follows: for KAP1, 5′-GATCCCTGGAGCCCCCATGGCCAGCCCAGTTCAAGAGACTGGGCTGGCCATGGGGGCTCCATTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAATGGAGCCCCCATGGCCAGCCCAGTCTCTTGAACTGGGCTGGCCATGGGGGCTCCAGG-3′; for the control, 5′-GATCCCTCCTCTATTATCAGTGGATATGTTTCAAGAGAACATATCCACTGATAATAGAGGATTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAATCCTCTATTATCAGTGGATATGTTCTCTTGAAACATATCCACTGATAATAGAGGAGG-3′. pTER+ was cut with BglII and HindIII, and the pTER backbone was purified from the gel. Sense and antisense oligos (100 pmol of each) were annealed in 50 μl of annealing buffer (10 mm potassium acetate, 3 mm Hepes-KOH, pH 7.4, and 0.2 mm magnesium acetate). One microliter of this oligos mixture and 100 ng of purified pTER+ backbone were used per ligation. Clones expressing pTER+-shKAP1 were selected with ampicillin resistance. To test the effect of sh-KAP1 knockdown, MCF-7 cells were co-transfected with p21-Luc, Tet-repressor plasmid pCDNA6/TR (Invitrogen), and pTER+-sh-KAP1 or pTER+-sh-Control. At 24 h post-transfection, cells were treated with 2 μm doxycycline for 4 or 24 h to induce the expression of their respective short hairpin RNA. Then the luciferase activity was measured and normalized against pRL-TK. pCDNA4-TO-Luc (Invitrogen) was used as positive control for doxycycline induction. Statistical Analysis—The error bar represents the S.D. of the mean. Statistical analyses were performed using one-way analysis of variance followed by post hoc comparisons based on a modified Newman-Keuls-Student procedure with p < 0.05 considered significant. Where appropriate, unpaired Student's t tests were also performed to determine the difference between two data groups. Differential Activation of ATM and ATR in Response to Dox and UV Treatment—We have recently demonstrated that the transcription of p21WAF1/CIP1 is distinctly regulated by KAP1 sumoylation status; sumoylation-mimetic KAP1 (SUMO-1-KAP1, Fig. 1A, left panel) enhances transcriptional repression by increasing the methylation of histone His-3–Lys-9 and decreasing the acetylation of His-3–Lys-9 and -Lys-14, whereas sumoylation-defective KAP1 (KAP1(3K/R), Fig. 1A, left panel) relieves transcriptional repression in an opposite manner (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Here, we report that Dox-treatment also activated the transcription of the DNA damage response gene Gadd45α (growth arrest and DNA damage clone 45) in a way similar to that of p21, whereas transfected KAP1 repressed the Dox-induced Gadd45α transcription in MCF-7 cells (Fig. 1A, right panel, second lane 2 versus first lane). Consistent with a previous report (3Lee Y.K. Thomas S.N. Yang A.J. Ann D.K. J. Biol. Chem. 2007; 282: 1595-1606Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), sumoylation-mimetic SUMO-1-KAP1 abolished the ability of Dox to induce Gadd45α expression (Fig. 1A, right panel, third lane). Notably, a L306P mutation of SUMO-1-KAP1 (SUMO-1-KAP1(L306P); Fig. 1A, left panel), which disrupts the interaction between coil-coiled domain of SUMO-1-KAP1 and KRAB domain with ZBRK1, almost completely relieved the repressive effect by SUMO-1-KAP1 (Fig. 1A, right panel, second lane). Our results support the notion that sumoylation of KAP1 enhances KAP1's function as a transcriptional co-repressor to attenuate Gadd45α transcription. Because both ATM and ATR function as both the sensors for and activators of DNA break repair, we set to examine whether ATM, ATR, or both are involved in mediating Dox-induced relief of KAP1 transcriptional co-repressor function. ATM-deficient fibroblast cells (pEBS7) and their counterparts (YZ5, in which the expression of wild-type ATM is restored by stable transfection of ATM in pEBS7 cells) were employed to explore the mechanism underlying KAP1/ZBRK1-mediated gene expression regulation in response to Dox treatment. As shown in Fig. 1B, ATM was rapidly and transiently phosphorylated on Ser-1981 after Dox treatment in MCF-7 cells with comparable kinetics to that observed in ATM-proficient YZ5 cells. The ATM-deficient pEBS7 cells served as a control to ensure the validity of the antibody against Ser-1981-phosphorylated ATM (Fig. 1B). We next compared the inducibility of Gadd45α-Luc in response to UV and Dox treatment in pEBS7 and YZ5 cells. In ATM-deficient pEBS7 cells, the Gadd45α transcription was induced by UV radiation (Fig. 1C, lanes 5–7), consistent with the idea that UV-induced DNA damage responses are mainly not ATM-dependent. Notably, Dox-induced Gadd45α transcription in pEBS7 cells was modest (Fig. 1C, lane 13), implying that ATM is required for Dox-mediated transcriptional activation. By contrast, Dox treatment induced Gadd45α-Luc activation (Fig. 1D, lane 13 versus lane 5), which was compromised by the expression of SUMO-1-KAP1 in a dose-dependent manner (Fig. 1D, lanes 14 and 15 versus lane 13) in ATM-proficient YZ5 cells. Although UV radiation activated a comparable induction in Gadd45α transcription (Fig. 1D, lanes 1 and 5 versus lanes 9 and 13), SUMO-1-KAP1 conferred a negligible effect on UV-induced Gadd45α promoter activation in YZ5 cells (Fig. 1D, lanes 6 and 7). Collectively, our results suggest that Dox and UV radiation induce Gadd45α transcription in distinct manners, and the sumoylation-mimetic SUMO-1-KAP1 only represses ATM-mediated Gadd45α transactivation. GK41 cells, human osteosarcoma U2OS cells stably transfected with doxycycline-inducible ATR-kd, were used to assess the role of ATR in KAP1-dependent Gadd45α transcriptional de-repression by Dox exposure. As shown in Fig. 1E, Dox treatment induced Gadd45α transcription by 2.4-fold (lane 4 versus lane 1), and the expression of SUMO-1-KAP1 repressed both basal (lanes 2 and 3) and Dox-inducible Gadd45α transcription (lanes 5 and 6) in the absence of doxycycline (such that the expression of ATR-kd is negligible) in GK41 cells. The expression of ATR-kd (by adding doxycycline (1 μg/ml) to medium) did not affect the basal and Dox-inducible Gadd45α transcription under the same experimental protocol (Fig. 1E, lanes 7 and 10–12 versus lanes 1 and 4–6). Taken together, we conclude that Dox treatment induces Gadd45α transcriptional activation via an ATM-dependent pathway to relieve KAP1-mediated trans-repression. However, it appears that SUMO-1-KAP1 had a lesser effect in repressing Dox-induced Gadd45α transcription in GK41 cells than in YZ5 cells. The exact mechanism underlying this discrepancy remains to be elucidated. Dox Treatment Induces KAP1 Phosphorylation at Ser-824 via ATM—To further examine the role of KAP1 Ser-824 phosphorylation in governing KAP1 function, we next engineered KAP1 mutants containing S824A or S824D substitutions. The S824D mutant was expected to mimic Ser-824-phosphorylated KAP1, whereas the S824A mutant reflected non-phosphorylated KAP1. By using KAP1(wt)-, KAP1(S/A)-, or KAP1(S/D)-transiently transfected MCF-7 cells, we then confirmed the specificity of antibody for Dox-induced KAP1 Ser-824 phosphorylation (Fig. 2A, left panel, lanes 4 and 7). The anti-KAP1-Ser-824 antibody has been used by Ziv et al. (21Ziv Y. Bielopolski D. Galanty Y. Lukas C. Taya Y. Schultz D.C. Lukas J. Bekker-Jensen S. Bartek J. Shiloh Y. Nat. Cell Biol. 2006; 8: 870-876Crossref PubMed Scopus (552) Google Scholar) to demonstrate that ATM phosphorylates KAP1 at Ser-824 in response to double-strand breaks. By using the level of tubulin to normalize for equal loading and the level of KAP1 to normalize for transfection efficiency, the quantitative analysis of Dox-induced KAP1 Ser-824 phosphorylation in MCF-7 cells was summarized in Fig. 2A (right panel). The phospho-Ser-824 signals detected in KAP1(S/A)- and KAP1(S/D)-transfected cells after treatment with Dox (1 μm) for 3 h represented the Ser-824-phosphorylated endogenous KAP1 (Fig. 2A, left panel, lanes 8 and 9). Our previous data suggest that ATM may be the key phosphatidylinositol 3-kinase protein-like kinase that induces Gadd45α transcriptional de-repression in Dox-treated cells, as Dox-induced Gadd45α-Luc activity was significantly reduced in ATM-deficient pEBS7 cells (Fig. 1C) as compared with that of ATM-complemented YZ5 cells (Fig. 1D). The possible role of ATM in mediating Dox-induced KAP1 Ser-824 phosphorylation was further analyzed in pEBS7 and YZ5 cells. Western analyses showed that Dox-induced endogenous and transfected KAP1 phosphorylation at