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Functional characterization of roles of histone deacetylases in the regulation of DNA damage response

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Functional characterization of roles of histone deacetylases in the regulation of DNA damage response
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Yuan, Zhigang
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University of South Florida
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SIRT1
NBS1
HDAC9
ATDC
p53
Protein acetylation
Dissertations, Academic -- Biochemistry and Molecular Biology -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
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Abstract:
ABSTRACT: Histone deacetylases (HDACs) are a family of enzymes whose functions have been overwhelmingly associated with gene expression and chromatin dynamics by modifying the histone tails. In recent years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activities have been found to be regulated by posttranslational acetylation on the ε-amino group of lysines. Here, we report that two DNA repair factors including NBS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Complementing) are acetylated proteins. SIRT1 could maintain NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
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Includes bibliographical references.
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by Zhigang Yuan.
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Includes vita.

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Functional Characterization of Roles of Histone Deacetylases in the Regulation of DNA Damage Response by Zhigang Yuan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Co-Major Professor: Edward Seto, Ph.D. Co-Major Professor: Peter Medveczky, M.D. Jiandong Chen, Ph.D. Burt Anderson, Ph.D. Date of Approval: September 13, 2007 Keywords: SIRT1, NBS1, HDAC9, ATDC, p53, protein acetylation Copyright 2007, Zhigang Yuan

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ACKNOWLEDGEMENTS First of all, I would like to give thanks to my great advisor, Dr. Seto, for his research direction, his patience, and hi s generous financial support. I would also like to thank Dr. Xiaohong Zhan g, who taught me lots of research techniques and did great collaborative work. My gratitude also extends to other members of my dissertation committee, Dr. Medveczky, Dr Anderson, Dr. Chen and Dr. Harton. Thanks to Dr. Pross for supporting me to apply for the American Heart Association Predoctoral Fellowship. T hanks to Dr. Klein, Dr. Ugen and Dr. Solomonson for the help for my graduate study. Special thanks to Dr. Yang for taking the time to come and chai r my examination committee. I will not forget Sally Baker, Kathy Zahn, Betty Sellers, Susan Chapman, Helen Chen-Duncan, and Donna Blaha, who took ca re of all the administrative works and made my graduate student life much less st essful. I also would like to give thanks to my classmate and friend J oe Chou for his generous help in my graduate study and routine daily life. Thanks to the form er graduate student Lily Lu for her help in my graduate study. I appreciate the help from members of Dr. Setos lab, past and present, the predoctoral fellowship granted from Americ an Heart Association, the technical

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support received from the Moffitt core fac ilities, and the help fr om other internal and external labs who generously provided pl asmids for facilitating my research. And, last but not least, my utmost grat itude goes to my family, my mom and dad, my mother-in-law and father-in-law, my brothers and sisters, for their love, support and taking care of my little son. Especial thanks to my wife Huayan Hou for her understanding and unwavering support.

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i TABLE OF CONTENTS List of Figures iv Abstract vi Section I: Sirt1 Regulates the Function of the Nijmegen Breakage Syndrome Protein 1 Abstract 1 Introduction 2 Materials and Methods 6 Results 11 NBS1 is an Acetylated Protein 11 Class III HDACs Regulate NBS1 Acetylation Level 12 NBS1 Interacts with SIRT1 15 SIRT1 Deacetylates NBS1 17 Acetylation Inhibits NBS1 Phosphorylation 19 Presence of SIRT1 Affects NBS1 phosphorylation 22 Acetylation of NBS1 Affects RDS 22 Acetylation of NBS1 Affe cts Cell Survival 26 Discussion 28 References 32

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ii Section II: Histone Deacetylase 9 Modulates Cell Survival by Regulating the AT DC-p53 Complex 42 Abstract 42 Introduction 43 Materials and Methods 45 Results 50 Immunoaffinity Purification of HDAC9 Containing Complex 50 ATDC Interacts with HDAC9 51 ATDC is an Acetylated Protein 54 ATDC Acetylation is Regul ated by HDAC9 54 HDAC9 Negatively Regulates AT DC Biological Function 58 ATDC Antagonizes p53-medi ated Apoptotic Pathway Activated by -irradiation and Forms a Physical Co mplex with p53 60 ATDC Inhibit p53 Function by Sequestering p53 into the Cytoplasm 64 HDAC9 Regulates the ATDC-p53 Complex Formation and Function 66 ATDC K116 Acetylation is the Major Functional Acetylation Site Responsible for Regulating p53 Interaction 68 ATDC is Overexpressed in Pancr eatic and Cervical Carcinoma 70 Growth Stress Induces ATDC Acet ylation 75 Discussion 78 References 81

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iii About the author End Page

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iv LIST OF FIGURES Figure 1. NBS1 is Acetylated 13 Figure 2. SIRT1 Interacts With N BS1 16 Figure 3. SIRT1 Deacetylates NBS1 18 Figure 4. NBS1 Acetylation Stat us Affects IR-induced NBS1 Phosphorylation 20 Figure 5. Presence of SI RT1 Affects NBS1 Ser343 Phosphorylati on 23 Figure 6. NBS1 Acetylation A ffects Radioresistant DNA Synthesis (RDS) 25 Figure 7. NBS1 Acetylation Affects Cell Survival 27 Figure 8. Immuno-affinity Purification of HDAC9 Containing Complex 52 Figure 9. ATDC is an Acetyl ated Protein Which is Regulat ed by HDAC9 55 Figure 10. ATDC Acetylation is Pr eferentially Regulated by HDAC9 59 Figure 11. HDAC9 Negatively Re gulates ATDC Biological Function 61 Figure 12. ATDC Facilitates Cell Survival by Antagonizing p53-mediated Apoptotic Pathway Activated by -irradiation and ATDC Forms a Physical Complex with p53 63 Figure 13. ATDC is a Cytoplasmic Prot ein 65 Figure 14. ATDC Inhibits p53 Function 67 Figure 15. HDAC9 Regulates the ATDC-p53 Complex 69

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v Figure 16. ATDC K116 Acetylation Play a Major Role in Regulating ATDC-p53 Interaction 71 Figure 17. Endogenous ATDC in Cervical Carcinoma Cells Regulates p53 Nuclear Localization and Function 74 Figure 18. Growth Stress Regulates ATDC Acetylation 76

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vi FUNCTIONAL CHARACTERIZATION OF ROLES OF HISTONE DEACETYLASES IN THE REGULA TION OF DNA DAMAGE RESPONSE ZHIGANG YUAN ABSTRACT Histone deacetylases (HDACs ) are a family of enzymes whose functions have been overwhelmingly associated with gen e expression and chromatin dynamics by modifying the histone tails. In rece nt years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activiti es have been found to be regulated by posttranslational acetylation on the -amino group of lysines. Here, we report that two DNA repair factors including N BS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Comp lementing) are ac etylated proteins. SIRT1 could maintain NBS1 in a hypoac etylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.

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1 SECTION I SIRT1 REGULATES THE FUNCTION OF THE NIJMEGEN BREAKAGE SYNDROME PROTEIN Abstract MRE11-RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor and is critical in regulating cellular responses to DNA double-strand breaks. NBS1, which is mutated in the human genetic disease Nijmegen break age syndrome, serves as the regulatory subunit of MRN. Phosphorylation of NBS1 by the ATM kinase is necessary for both activation of the S-phase checkpoint and for efficient DNA damage repair response. Here, we report that NBS1 is an acetylated protein and that the acetylation level is tightly regulated by the SIRT1 deacetylase. SIRT1 associates with the MRN complex and, im portantly, maintains NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. Our result s demonstrate the presence of crosstalk between two different posttranslational modifications in NBS1 and st rongly suggest that deacetylation of NBS1 by SIRT1 plays a ke y role in the dynamic regulation of the DNA damage response and in the ma intenance of genomic stability.

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2 Introduction Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition of chromosomal instability that is clinic ally manifested by symptoms including microcephaly, a distinct facial appearance, growth retardation, immunodeficiency, radiation se nsitivity, and a strong pr edisposition to lymphoid malignancy (van der Burgt et al., 1996; Shiloh, 1997; Digweed and Sperling, 2004). Cells from NBS patients exhibit radi ation hypersensitivity, radioresistant DNA synthesis (RDS), chromosomal instability, and cell cycle checkpoint defect (Tauchi et al., 2002). Mutati ons in NBS1 (also known as nibrin or p95), the product of the Nijmegen breakage syndrom e gene, are responsible for NBS (Varon et al., 1998). The N-terminus of NBS1 protein contains a forkheadassociated (FHA) domain adjacent to a breast cancer carboxy-terminal (BRCT) domain, both of which are commonly found in cell cycle c heckpoint proteins. The C-terminus of NBS1 is required for induction of MRN complex-mediated apoptosis in response to irradiation (Stracker et al., 2007). The ATM protein kinase, a multi-ta sking DNA damage sensor, is mutated in individuals with the radiosensitivity di sorder ataxia-telangiectasia. Following cellular exposure to ionizing r adiation (IR), ATM undergoes rapid autophosphorylation at Ser1981, resulting in the conv ersion of the inactive dimer form to active monomers (Bakkenist and Kastan, 2003). Activated ATM phosphorylates a number of cellular substrates including NBS1 (Lim et al., 2000; Wu et al., 2000; Zhao et al., 2000). NBS1 associates with MRE11 and RAD50 to form a protein complex (MRN complex) involved in detection, signaling, and

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3 repair of DNA damage. Using an Nbs1 k nockout cell line, NBS1 was shown to be essential for homologous recombinat ion DNA repair in vertebrate cells (Tauchi et al., 2002). Although phosphorylation of NBS1 does not affect MRN association, this modifi cation is functionally impo rtant since mutant NBS1 (S343A) cannot completely complement radiosensitivity in cell lines lacking functional NBS1 (NBS cells) (G atei et al., 2000; Lim et al., 2000; Zhao et al., 2000). In addition to serving as a downs tream effector of ATM, NBS1 may function in activating ATM (Cerosaletti et al., 2006; Lee and Paull, 2005; You et al., 2005). In fact, NBS1 phos phorylation may be required for activation of the Sphase checkpoint by stimulating ATM-mediated phosphorylation of Chk2 (Lee and Paull, 2004). Besides phosphorylation, the functions and activities of an increasing number of proteins hav e been found to be regulated by posttranslational acetylation on the -amino group of lysines (Glozak et al., 2005; Kouzarides, 2000; Yang, 2004). This modification prev ents positive charges from forming on the amino group of lysines and, as a result, has a significant impact on the electrostatic properties of the protein. Over thirty proteins have been reported to possess lysine acetyltransferase activity, and many of these enzymes were first thought to specifically ac etylate histones, but later were found to have a wide range of protein substrates in addition to histones (Sterner and Berger, 2000; Roth et al., 2001; Yang, 2004). Also, m any acetyltransferases, including p300, CBP (CREB-binding protein), and PCAF (p300/CBP-associated factor) are transcriptional co-activators.

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4 Like many covalent protein modi fications, posttranslational lysine acetylation is highly reversible, and increasing evidences suggest that acetylation/deacetylation, lik e phosphorylation, is import ant in the regulation of a number of biological processes (Kouzar ides, 2000). Thus, in order to fully understand the pathways that modulate the functions of NBS1, it is important to determine whether NBS1 undergoes acet ylation and if so, whether this modification is reversibly regulated by deacetylation. Histone deacetylases (HDACs) are enzym es that catalyze the removal of acetyl moieties from the -amino groups of conserved lysine residues in the amino terminal tail of histones. The re moval of this modification strengthens histone-DNA interactions and may generate specific docking surfaces for proteins that regulate chromatin foldi ng and/or transcription. Results from numerous studies overwhelmi ngly support the prediction that HDACs play crucial roles in gene transcription and most likel y affect all eukaryotic biological processes that involve chromatin. Re cent studies have shown that many nonhistone proteins can serve as HDAC s ubstrates and many HDACs, like histone acetyltransferases (HATs), regulate impor tant biological processes that extend beyond histones and gene transcription (Glozak and Seto, 2005). In humans, HDACs are divided into th ree categories: the class I RPD3like proteins (HDAC1, HDAC2, HDAC3, HD AC8); the class II HDA1-like proteins (HDAC4, HDAC5, HDAC6, HDAC7, HDAC 9, and HDAC10); and the class III Sir2-like proteins (SIRT1, SIRT2, SIRT 3, SIRT4, SIRT5, SIRT6, and SIRT7). The class III proteins do not exhibit any sequence similarity to the other HDAC

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5 family members and differ from the other HDACs in that they require the cofactor NAD + for activity. Whereas the NAD + -dependent class III deacetylases are specifically inhibited by nicotinamide, class I and II HDACs are specifically s ensitive to the inhibitor tricohostatin A (TSA) Of the seven human Sir2-like proteins (s irtuins), SIRT1 is most similar to the yeast Sir2 protein, wh ich is the prototypic cla ss III HDAC (Guarente, 2006; Blander and Guarente, 2004). In vitro, SIRT1 preferentially deacetylates histones H4K16 and H3K9, interacts with and deacetylates histone H1 at K26, and may mediate heterochromatin formati on (Vaquero et al., 2004). Besides histones, more than a doz en nonhistone proteins have been found to serve as substrates for SIRT1 (Blander and Guar ente, 2004; Haigis and Guarente, 2006). SIRT1 regulates the tumor suppressor protein p53 and FOXO3 to suppress apoptosis and promote cell survival (B runet et al., 2004; Giannakou and Partridge, 2004; Luo et al., 2001; Vaziri et al., 2001). SIRT1 plays a role in several biological processes includi ng stress resistance, metabolism, differentiation, and aging (Haigis and Guar ente, 2006). In addition, thymocytes derived from mice lacking SIRT1 ex hibit increased sensitivity to -irradiation (Cheng et al., 2003). In the present study, we show that the NBS1 protein is acetylated. Furthermore, we found that SIRT 1 binds to and deacetylates NBS1 in vitro and in vivo and, importantly, maintains NBS1 in a hypoacetylated state that is required for IR-induced NBS1 phosphorylation. Induction of NBS1 hyperacetylation greatly reduces NBS1 phosphorylation. Consistent with the importance of

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6 hypoacetylated NBS1 in DNA damage repai r, cell survival decreases and RDS increases in NBS1-deficient cells co mplemented with hyper acetylated NBS1. Together, our findings strongly implic ate crosstalk between two different posttranslational modifications in the functi on of NBS1. Additionally, our results uncovered NBS1 as a substrate for SIRT1 and provide convincing evidence that SIRT1 acts upstream of NBS1. Materials and Methods Plasmids, Antibodies, and Viruses The following expression plasmids us ed in these experiments have been described previously: pcDNA3-Myc-NBS1 (Maser et al., 2001), HA-NBS1 (Lim et al., 2000), Flag-ATM (Lim et al., 2000), Fl ag-PCAF (Yang et al., 1996), HA-p300 (Aizawa et al., 2004), pRc/RSV-HA-C BP (Zhang et al., 2000), Flag-SIRT1-7 (Michishita et al., 2005; North et al., 2003), Myc-SIRT1 (Langley et al., 2002), GST-SIRT1 (Langley et al., 2002), and Myc-SI RT1(H363Y) (Langley et al., 2002). The plasmid encoding Myc-NBS1mt was generated using the QuickChange SiteDirected Mutagenesis kit following the manufacturers protocol (Stratagene). Plasmids encoding GST-NBS1 deletion mutants were created by inserting PCR products of NBS1 fragments into Bam H1/Not 1 digested pGEX-5X-1 vectors (Amersham). pBS/U6-SIRT1 was constructed by inserting oli godeoxynucleotides, which targeted the sequence 5GAAGTTG ACCTCCTCATTGT3 into the pBS/U6 vector (Sui et al., 2002). SIRT1 siRNA and control siRNA adenoviruses were described previously (Rodgers et al., 2005). Plasmids that express Myc-tagged

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7 NBS1 lysine to glutamine mutants (5KQ: K544Q/K665Q/K690Q/K698Q/K715Q; 7KQ: K441Q/K504Q/K544Q/K665 Q/K690Q/K698Q/K715Q; 9KQ: K233Q/K334Q/K441Q/K504Q/K544 Q/K665Q/K690Q/K698Q/K715Q; 10KQ: K208Q/K233Q/K334Q/K441Q/K504 Q/K544Q/K665Q/K690Q/K698Q/K715Q) were generated by standard PCR and subcloning. Mouse affinity purified monoclonal anti-Flag M2, rabbit affinity purified polyclonal anti-HA, and mouse monoclonal anti-acetylated-t ubulin (clone 6-11B-1) antibodies were purchased from Sigma. Mouse monoclonal anti-c-Myc (clone 9E10) and mouse monoclonal anti-p53 (cl one DO-1) antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-hNBS1 rabbit polyclonal anti-phosphorylated-Ser343hNBS1, rabbit polyclonal anti-phosphorylatedSer343-mNbs1, and rabbit polyclonal ant i-ATM were purchased from Novus Biologicals. Mouse monoclo nal anti-hNBS1 (clone 34) was purchased from BD Biosciences. Protein A pur ified mouse monoclonal ant i-phosphorylated-Ser1981ATM (clone 10H11.E12) was purchased from Rockland Immunochemicals. Mouse monoclonal anti-hNBS1 (clone 1C3), mouse monoclonal anti-MRE11 (clone 12D7), and mouse monoclonal antiRAD50 (clone 13B3) were purchased from GeneTex. Rabbit polycl onal anti-acetyl-lysine, rabbit polyclonal anti-SIRT1, and mouse monoclonal anti-GST (clone DG122-2A7) antibodies were purchased from Upstate (Millipore). Mouse monoclonal anti-BrdU (clone BMC9318) was purchased from Roche. Rabbit polyclon al anti-acetylated-Lys382-p53 was purchased from Cell Si gnaling Technology.

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8 Cell Culture, Transfection, and Adenovirus Infection Sirt1 +/mice were time-mated to screen for homozygous Sirt1 -/mutant embryos by PCR. MEFs were gener ated from 13.5 dpc embryos using standard methods. HeLa, 293T, and murine Sirt1 +/+ and Sirt1 -/fibroblasts were grown in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (pen/stre p). The NBS cell line (GM07166) was obtained from Coriell Cell Repository and grown in minimum essential medium (MEM) with 10% FCS and pen/strep. All transfections were normalized with equal amounts of parental ve ctor DNA. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. To generate stable cell lines, NBS cells (GM 07166) transfected with vector alone or various expression plasmids were grown in the presence of G418 (200 mg/ml, for cells that received NBS1 expression pl asmids) and puromycin (0.5 mg/ml, for cells that received PCAF expression plasmi ds) for 10 days. Resistant colonies were isolated, pooled together, and grown for further analysis. For viral infection, HeLa cells were infected with adenovirus for 24 h in DMEM with 0.5% BSA as previously described (Rodgers et al., 2005). Immunoprecipitation and Western Blot Analysis For immunoprecipitations, cells were lyse d in buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% NP-40, and protease inhibito r cocktail) containing either 500 mM NaCl (high stringency) or 150 mM NaCl (l ow stringency). The lysates were incubated with the primary antibody overnight at 4 o C. The resultant

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9 immunocomplexes were collected, was hed four times in lysis buffer, and resolved by SDS-PAGE. For immunoblotting, samples were transferred onto nitrocellulose membranes. Membranes were probed with the appropriate antibodies. Proteins of interest we re visualized using the Chemiluminescent Detection Kit (Pierce). Ion Trap Mass Spectrometry 293T cells were transfected wit h the Myc-NBS1 expressi on plasmid and treated with TSA (1.3 M ) and nicotinamide (20 mM) overnight. The cells were then lysed in high stringency buffer containi ng 10 mM NaB and 10 mM nicotinamide. Cell extracts were subjected to imm unoprecipitation with ant i-Myc antibodies. The immune complexes were resolved by SDS-PAGE and stained with colloidal Blue (Invitrogen). The Myc-NBS1 s equence was analyzed with an in-house algorithm (EnzOpt) for a dual enzyme strategy maximizing proteotypic peptide coverage of all lysines. The appropriate Myc-NBS1 gel band was excised and divided into two parts. Each part wa s subjected to in-gel reduction and carboxyamidomethylation followed by separat e tryptic or chymotryptic digestion. Acetylated peptides fr om each digest were detected and sequenced using microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ linear quadrupole ion trap mass spectrometer. Data analysis was facilit ated with SEQUEST and the Proteomics Browser Suite (Thermo).

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10 GST Pull-Down Assay GST and GST-NBS1 deletion mutants were expressed and purified from bacteria using standard methods. Equimolar quantit ies of the various purified proteins were conjugated to glutathione-Sephar ose beads and incubated with HeLa whole cell lysates for 1 h at 4C. After extens ive washing, bound prot eins were eluted and analyzed by Western blotting with anti-SIRT1 antibodies. In Vitro Deacetylation Assay In vitro deacetylation of NBS1 by SIRT 1 was performed using an approach similar to that used previously for detection of PGC-1 acetylation (Nemoto et al., 2005). RDS Assay The RDS assay was performed as descr ibed previously (Zhao et al., 2000) with minor modifications. Briefly, NBS cells were transfected with either pEGFP-C3 (Clontech) or pEGFP-C3 and the Myc-N BS1 expression plasmid at a ratio of 1:10. In some experiments, cells were also transfected with an additional plasmid encoding Flag-PCAF. Cells were -irradiated with 15 Gy, incubated for 1 h at 37 o C, and then incubated for an additional 2 h in a medium containing 100 M of BrdU. BrdU incorporation was detected using an anti -BrdU antibody following immunostaining procedures descr ibed previously (Zhang et al., 2004). Alternative RDS assays were performed as described by Lim et al. (2000) and by Zhao et al. (2002). Briefly, cells were labeled for 24 h in medium

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11 containing 10 nCi/ml of 14 C-thymidine. The cells were washed once with PBS and then incubated for 24 h in a nonradioactiv e medium. After treatment with IR or left untreated, cells were incubated at 37C for 30 min and pulse-labeled for 30 min in a medium cont aining 2.5 mCi/ml of 3 H-thymidine. Cells were then washed with PBS and lysed with 0.5 ml of 0.2 M Na OH. Radioactivity was quantified in a liquid scintillation counter, and the resulting ratios of 3 H/ 14 C were calculated and compared with ratios of non-irradiated cells. Colony Survival Assay Colony survival assays were performed as previously described (Zhao et al., 2002) with minor modifications. Briefly, NBS cells were plated in quadruplicate (1,000 cells per 60-mm tissue cult ure dish). The cells were -irradiated with 0, 2, or 5 Gy. After two wee ks, dishes were washed with PBS, fixed in ice-cold methanol for 15 min, and then st ained with Giemsa stain for 30 min. Colonies on each plate were quantified and expressed as the percentage of the unirradiated control. Results NBS1 is an Acetylated Protein The NBS1 protein (accessi on BAA28616) contains three lysine acetylation consensus motifs, Kx 1-2 x/KK (Yang, 2004), at resi dues 233-237 (KGHKK), 683687 (KNFKK), and 686-690 (KKFKK). Thus in addition to phosphorylation, NBS1 is potentially modified by posttranslati onal lysine acetylation. To determine if NBS1 is indeed a substrate for ac etyltransferases, 293T cells were co-

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12 transfected with plasmids expressing a Myc-tagged NBS1 protein and three different acetyltransferases (PCAF, p300, or CBP). Acetylated NBS1 was readily detected in cells that overexpressed PCAF or p300 but not in cells that overexpressed CBP (Figure 1A). The le vel of NBS1 acetylation by PCAF was dose-dependent (Figure 1B). Analysis of pur ified Myc-NBS1 by LC tandem mass spectrometry (LC-MS/MS) revealed that 10 of t he 70 lysine residues were acetylated. These included K208, K233, K334, K441, K504, K544, K665, K690, K698, and K715 (Figure 1C). Mutation of all ten sites from lysine to arginine resulted in a dramatic decrease in the overall NBS1 acetyl ation levels as determined by Western blot analysis with anti-acetyl-l ysine antibodies (Figure 1D). A small amount of residual acetyl ation can still be detected by Western blotting with anti-acetyl-lysine antibodies, suggesting that there could be minor acetylation sites that esc aped mass spectrometry analysis. Class III HDACs Regulate NBS1 Acetylation Level To examine whether NBS1 might be regulated by deac etylation, we immunoprecipitated Myc-NBS1 from 293T cells treated with HDAC inhibitors. As shown in Figure 1E, treatment of cells wit h TSA, a class I and II HDAC inhibitor, did not affect the acetylat ion status of NBS1. In contrast, cells treated with nicotinamide, a class III (Sir2 family) HD AC inhibitor, resulted in a notable increase in acetylation of NBS1. Treatment with a combination of TSA and nicotinamide was not associated with any ad ditional increase in NBS1 acetylation

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A B 13 Figure 1. NBS1 is Acetylated (A) 293T cells were co-transfected with equal amounts (4ug) of Myc-NBS1 one of the following expression plasmids: Flag-PCAF, HA-p300, or HA-CBP. Cell lysates were immunoprecipitated (IP) under high stringency conditions using antiMyc-NBS1 + + + + CBP + p300 + PCAF + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc Myc-Ac-NBS1 Myc-NBS1 IB anti-AcK382-p53 IB anti-p53 AcK382-p53 p53 IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc Myc-NBS1 + + + + ++ +++ Flag-PCAF + IB anti-Flag D IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-Flag Myc-NBS1mt + + Myc-NBS1 Flag-PCAF + + + + 69 C 20 23 33 44 50 54 66 69 71 FH BRC MI AI E Myc-Ac-NBS1 Myc-NBS1 + + + + Nicotinamide TSA + + + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc G Nicotinamide + Myc-NBS1 IB anti-Ac-tubulin Ac-tubulin IB anti-Ac-H4K16 IB anti-H4 Ac-H4K16 histone H4 IB anti-AcK IB anti-NBS1 IP anti-NBS1 Mock IP + + + F Myc-NBS1mt + + Myc-NBS1 Nicotinamide + + + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc Myc-Ac-NBS1(wt/mt) Myc-NBS1 (wt/mt) + + + + + +

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14 Myc antibodies. Immunoprecipitates were subjected to We stern blot (IB) analysis using anti-acetyl-lysine (AcK) antibodies. The blot was stripped and re-probed with anti-My c antibodies to confirm equal immunoprecipitation efficiency and loading. As CBP (but not PCAF) is known to acetyl ate p53-K382, a Western blot was performed with anti-AcK382p53 to confirm that the expressed CBP was functional. (B) 293T cells were co-transfected with Myc-NBS1 (4 ug) and Flag-PCAF (0, 0.5, 1, or 4 ug). Cell lysates were subjec ted to immunoprecipitation/Western blot analysis using the indicated antibodies. Western blot analysis of whole extracts was performed to assess PCAF expression (bottom panel). (C) Myc-NBS1, which was expressed and purified from 293T ce lls, was digested with trypsin and subjected to ITMS. Positions of the unambiguously-identified acetylated lysine residues are shown. FHA, forkhead-associated domain; BRCT, BRCA1 C-terminal domain; MIR, MRE11-interacting domai n; AIR, ATM-interacting domain. (D) 293T cells were co-transfected with Myc-NBS1 (wild-type or lysine to argini ne mutant [mt]) and Flag-PCAF (4 ug each), as indicated. Total and acetylated NBS1 levels were analyzed by immunoprecipitation/Western blotting. (E) 293T cells were transfected with plasmids that expressed Myc-NBS1 (4 ug). Twenty-four hours post-transfection, cells were left untreated or treated overnight with TSA (1.3 uM), nicotinamide (20 mM), or TSA plus nicotinamide. Cell lysates were subjected to immunoprecipitation/Western blotting. W hole cell extracts were probed with anti-Ac-tubulin and anti-Ac-H4K16 to confirm that TSA is functional under the treatment condition (lower panels). (F) 293T cells were transfected with plasmids (4 ug each) expressing wild-type or mutant Myc-NBS1 as indi cated and then treated with 20 mM nicotinamide overnight. MycNBS1 acetylation and protein expression were exami ned by immunoprecipitation/Western blotting. (G) HeLa cells were treated with nicotinamide (20 mM) or left untreated overni ght. Cell lysates were then immunoprecipitated under high stringency conditions with monoclonal anti-NBS1 antibodies. Endogenous acetylated NBS1 was analyzed by Western blotting with a mixture of polyclonal anti-acetyl-lysine antibodies. when compared to treatment with nicoti namide alone. Thus, NBS1 might be deacetylated by class III HDACs but not by cl ass I or II HDACs. Hyperacetylation of NBS1 is specific to nicotinamide treatment since the NBS1-acetylation mutant did not respond to this HDAC inhibitor (F igure 1F). Anti-acetyl-lysine Western blot analysis of anti-NBS1 immunoprecipitates obtained under high stringency conditions in which neither MRE11 no r RAD50 co-precipitated with NBS1, revealed that nicotinamide enhanced the acetylation state of endogenous NBS1 (Figure 1G), providing proof that endoge nous NBS1 is regulated by class III HDACs.

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15 NBS1 Interacts with SIRT1 An interesting feature of the class II I HDACs is their subcellular location (Michishita et al., 2005). SIRT1, SIRT6, and SIRT7 ar e present in the nucleus while SIRT2 is located in the cytosol despite the possibility of deacetylating H4K16 during mitosis (Vaquero et al., 2006) SIRT3, SIRT4, and SIRT5 are localized in the mitochondria. Of the three nuclear SIRTs, SIRT1 has been shown to possess robust deacetylase enzymatic activity, while SIRT6 and SIRT7 have been reported to have very weak or no deacetylase activity (Liszt et al., 2005; North et al., 2003). Therefore, SIRT1 is lik ely responsible for the deacetylation of NBS1. Since most, if not all, deacetylase subs trates interact with their respective enzymes, we first tested the ability of NBS1 to interact with SIRT1. As shown in figure 2A, Flag-SIRT1 co-precipitated with Myc-NBS1. Similarly, Myc-NBS1 coprecipitated with Flag-SIRT1 (Figure 2B). The SIRT1-NBS1 interaction is highly specific since none of the other Sir2 family members (SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7) co-precipita ted with NBS1 under id entical conditions (Figure 2C). This association of SIRT1 and NBS1 also occurs in the absence of overexpression, epitope tagging of either protein, or IR treatment (Figure 2D). Because the subunits of the MRN comple x most likely do not act individually, MRE11 and RAD50 also co-precipitated wit h SIRT1 as expected, suggesting that SIRT1 associates with the whole MRN co mplex. Analyses of three different GST-NBS1 deletions indicat ed that SIRT1 interacts with the C-terminus of NBS1

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A C + Input Flag-SIRT1 + 16 Figure 2. SIRT1 Interacts With NBS1 (A, B, C) 293T cells were co-transfected with plasmids (4ug each) encoding the indicated Myc and Flag fusion proteins. Anti-Myc and anti-Flag immunoprecipitates obtained under low stringency conditions were analyzed by Western blotting with the indicated antibodies. (D) HeLa cells were irradiated (10 Gy) or left untreated. One hour after irradiation, endogenous SIRT1 was immunoprecipitated under low stringency conditions with anti-SIRT1 polyclonal antibodies. Flag-SIRT1 Myc-NBS1 IB anti-Flag IB anti-Myc Flag-SIRT1 + + + + IP anti-Myc + + Mock IP + Myc-NBS1 + + + Myc-NBS1 IB anti-Flag IB anti-Myc Flag-SIRT1 B + Input Myc-NBS1 + + + + IP anti-Flag + + Mock IP + Flag-SIRT1 + + + IP anti-Flag IB anti-Myc IP anti-Flag IB anti-Flag IB anti-Myc Myc-NBS1 + + + + + + + + Flag-SIRT3 Flag-SIRT2 + + Flag-SIRT4 + Flag-SIRT5 + Flag-SIRT6 + Flag-SIRT7 + GST-NBS1(572-754) GST-NBS1(226-578) D E GST-NBS1(1-230) IR + + + Mock IP + + + Input + IP anti-SIRT1 + + IB anti-NBS1 IB anti-MRE11 IB anti-RAD50 NBS1 input GST IB anti-SIRT1 MRE1 RAD50 IB anti-GST IB anti-SIRT1 SIRT1

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17 Immune complexes were analyzed by Western blotting with anti-NBS1, anti-MRE11, anti-RAD50, or anti-SIRT1 antibodies. For maximum clarity, the image gathered for t he left upper middle panel (MRE11, lanes 1 and 2) was underexposed compared to the right upper middle panel (MRE11, lanes 3-6). (E) GST-NBS1 deletion mutants coupled to Sepharose beads were incubated with whole HeLa cell extrac ts. After the beads were washed, bound proteins were eluted and analyzed by Western blotting with an anti-SIRT1 antibody. The blot was stripped and re-probed with anti-GST antibodies to confirm equal quantities of GST proteins in each reaction. (residues 572-754), a segment that contai ns the MRE11 and ATM interaction region (Figure 2E). SIRT1 Deacetylates NBS1 To test whether SIRT1 deacetylates N BS1, we examined the effect of RNAimediated SIRT1 knockdow n on NBS1 acetylation in 293T cells. BS/U6templated siRNA efficiently silenced ex pression of SIRT1 but not the control protein -actin, as monitored by Western blot analysis (Figure 3A). In agreement with the finding that SIRT1 binds NBS1, acetylation of both Myc-NBS1 (Figure 3A) and endogenous NBS1 (Figure 3B) was signi ficantly increased as a result of SIRT1 knockdown. Unlike knock-down of SIRT1, depletion of SIRT6 or SIRT7 did not affect NBS1 acetylation leve ls (Figure 3C). Furthermore, PCAFacetylated NBS1 was deacetylated by wild-t ype SIRT1 (Figure 3D), but not by a catalytic-defective SIRT1 mutant (H363Y ) (Figure 3D) or by SIRT2, SIRT3, SIRT4, SIRT6, SIRT7 (Figure 3E). In vitro deacetylation assays using purified SIRT1 in the presence of NAD + further confirmed that NBS1 is a substrate of SIRT1 (Figure 3F). Collectively, our data strongly suggest that SIRT1 modulates the acetylation status of NBS1 via its deacetylase activity.

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A B 18 Figure 3. SIRT1 Deacetylates NBS1 (A) 293T cells were co-transfected with equal amounts (4 ug) of Myc-NBS1 plasmid and either a plasmid encoding pBS/U6-SIRT1 or control siRNA. Western blots were performed with the indicated antibodies to assess the levels of SIRT1, -actin, total Myc-NBS1, and acetylated Myc-NBS1. (B) 293T cells were infected with either adenovirus that Myc-NBS1 + + + SIRT1 siRNA Control siRNA + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc Myc-Ac-NBS1 Myc-NBS1 IB anti-SIRT1 IB anti-actin SIRT1 -actin + + SIRT1 siRNA Control IP anti-NBS1 + + IB anti-AcK Ac-NBS1 IB anti-NBS1 NBS1 D Myc-SIRT1 + IP anti-HA IB anti-AcK IP anti-HA IB anti-HA IB anti-Flag HA-Ac-NBS1 HA-NBS1 Flag-PCAF + Flag-PCAF Myc-SIRT1(H363Y) + + + HA-NBS1 + + + C Control siRNA + IB anti-Myc Myc-SIRT1 WT/H363Y IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc Myc-Ac-NBS1 Myc-NBS1 SIRT6 siRNA SIRT1 siRNA + + Myc-NBS1 SIRT7 siRNA + + + + + E Flag-SIRT1 + IB anti-Flag IB anti--actin Flag-SIRT6 -actin Flag-SIRT7 SIRT6 siRNA + SIRT7 siRNA + + + Flag-SIRT6 Flag-SIRT7 + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-Flag Myc-Ac-NBS1 Myc-NBS1 Flag-PCAF Myc-NBS1 + + + + + + + Flag-SIRT3 Flag-SIRT2 + + Flag-SIRT4 + Flag-SIRT6 + Flag-SIRT7 + Flag-PCAF + + + + + + + F Flag-SIRT1 Nicotinamide + NAD + + +++ GST-SIRT1 + + + + Myc-Ac-NBS1 IB anti-AcK Flag-SIRT2/3/4/6/7 IB anti-Myc Myc-NBS1

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19 expresses control siRNA or adenovirus that expresses SIRT1 siRNA. Western blots were performed with the indicated antibodies to assess the acetylation of endogenous NBS1 and NB S1 immunoprecipitation efficiency. (C) Top two panels, 293T cells were co-transfected with equal amounts (4 ug) of Myc-NBS1 plasmid and eit her a plasmid encoding SIRT1 siRNA, SIRT6 siRNA, SIRT7 siRNA, or control siRNA. Wester n blots were performed with the indicated antibodies to assess the levels of total Myc-NBS1 and acetylated Myc-N BS1. Bottom two panels, similar experiments were performed with over-expression of Flag-SIRT6 and Flag-SIRT7 to sh ow that SIRT6 and SIRT7 siRNAs are functional. (D) 293T cells were co-transfected with plasmids that express HA-NBS1, FlagPCAF, and either a wild-type or a catalytically-defective Myc-SIRT1. Acetylation of HA-NBS1 and all protein levels were determined with direct Western blotting or immunoprecipitations followed by Western bl otting using the indicated ant ibodies. (E) 293T cells were co-transfected with plasmids that express Myc-NBS1, Flag-PC AF, and different Flag-tagged SIRTs. Acetylation of Myc-NBS1 and all protein levels were determined with direct Western blotting or i mmunoprecipitations followed by Western blotting using the indicated antibodies. (F) 293T cells were transfected with Myc-NBS1 and Flag-PCAF. Anti-Myc immunoprecipitates were incubated with recombinant GST-SIRT1 in the absence or presence of NAD + (lane 2, 1mM; lane 3, 10 mM) and nicotinamide (10 mM) at 30 o C for 1 h. Western blot analysis of acetylated Myc-NBS1 was then performed using antiacetyl-lysine (AcK) antibodies. Acetylation Inhibits NBS1 Phosphorylation The balance between acetylation and deacetylation may have functional consequences for NBS1. Previous work has shown that ATM-mediated phosphorylation of NBS1 at Ser343 in res ponse to DNA damage is critical for the DNA damage responses and for activation of the cell cycle S-phase checkpoint (Lim et al., 2000; Wu et al., 2000; Zhao et al., 2000). We hypothesized that different posttranslational modifications of NBS1 affect the phosphorylation of NBS1 at Ser343. NBS cells (GM07166) expressing Myc-NBS1 alone or in combination with Flag-PCAF were irradiated, and the time course of NBS1 Ser343 phosphorylation was examined via We stern blot analysis. As shown in Figure 4A, overexpression of PCAF, which acetylates NBS1, reduced the levels of NBS1 phosphorylation at all times ex amined. Similar results were obtained

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20 A pS343-Myc-NBS1 IB anti-pS343-NBS1 IB anti-Myc IB anti-Flag Myc-NBS1 Flag-PCAF IP anti-Myc IB anti-AcK Myc-Ac-NBS1 IB anti-pS1981-ATM IB anti-ATM pS1981-ATM ATM Myc-NBS1 + + + + + + + + + + + + + + Flag-PCAF + + + + + + + Post IR (min) 0 30 60 0 30 60 0 60 120 60 120 240 240 0 7 8 9 10 11 12 13 14 IB anti-pS343-NBS1 IB anti-Myc pS343-Myc-NBS1(wt/mt) Myc-NBS1(wt/mt) D Post IR (min) 0 30 60 0 30 60 0 30 60 0 30 60 Nicotinamide + + + + + + Myc-NBS1mt + + + + + + + + + Myc-NBS1 IB anti-pS343-NBS1 IB anti-Myc pS343-Myc-NBS1(wt/mt) Myc-NBS1(wt/mt) Post IR (min) 0 60 0 60 0 60 0 60 0 60 0 60 Myc-NBS1(wt) + + + + Myc-NBS1(5KQ) + + Myc-NBS1(7KQ) + + Myc-NBS1(9KQ) + + Myc-NBS1(10KQ) + + E IB anti-NBS1 IB anti-ATM NBS1 ATM C IP anti-NBS1 + + + + IR (6 Gy) + + Nicotinamide + + + Mock IP + IP anti-ATM + + Nicotinamide Post IR 0 30 60 0 30 60 Myc-NBS1 + + + + + + + + + IB anti-pS343-NBS1 IB anti-Myc IB anti-pS1981-ATM IB anti-ATM B IP anti-Myc IB anti-AcK

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21 Figure 4. NBS1 Acetylation Status Affects IR-induced NBS1 Phosphorylation (A) The NBS cell line GM07166 was transfected with equal amounts (4 ug) of Myc-NBS1 expression plasmid and FlagPCAF expression plasmid or empty vector Twenty-four hours post-transfection, ce lls were irradiated (6 Gy) or left untreated. Cell lysates were subjected to Western blot anal ysis or to high stringency imm unoprecipitation/Western blot analysis, as indicated. (B) NBS cells transfected with Myc-NBS1 expression plasmid (4 ug) were treated overnight with 20 mM of nicotinamide or left untreated. The cells were t hen irradiated (6 Gy) or left untreated. Cell lysates were prepared after the indicated time and subjected to direct Western blot analysis or high stringency immunoprecipitation/Western blot analysis using the indicated antibodies. (C) To examine the effect of nicotinamide on ATM-NBS1 interactions, ATM and NBS1 were immunoprecip itated with anti-ATM antibodies and anti-NBS1 antibodies, respectively, from extracts prepared fr om HeLa cells treated nicotinamide. NBS1 and ATM were identified in the immunoprecipitates by Western blotting with anti-NBS1 and antiATM, respectively. (D) NBS cells were transfected with plasmids encoding either wild-typ e or acetylation-defective Myc-NBS1 (4 ug). Parallel cultures were then irradiated or left unirradiated in the presence or absence of nicotinamide. Lysates were analyzed by Western blots with the indicated antibodies. (E) NBS cells were transfect ed with plasmids encoding either wild-t ype Myc-NBS1 or acetylation-mimic MycNBS1 mutants (4 ug). Parallel cultures were then irradiated or le ft unirradiated and lysates were analyzed by Western blots with the indicated antibodies. when NBS cells, complemented with Myc-N BS1, were treated with nicotinamide to induce NBS1 hyperacetylation (Figur e 4B). The decrease in NBS1 Ser343 phosphorylation was not due to a decrease in ATM protein levels (Figures 4A and 4B), a decrease in activated ATM (a s measured by phospho-S1981 of ATM; Figures 4A and 4B), or a decrease in ATM-NBS1 interaction (Figure 4C). Treatment with nicotinamide had no effe ct on NBS1 Ser343 phosphorylation in an NBS1 mutant that was refractory to acetylation/deacetylation (Figure 4D). To further examine the effect of acetylation/deac etylation on NBS1 Ser343 phosphorylation, we construc ted four plasmids that express NBS1 acetylationmimic mutants, with lysine to glutami ne changes, and introduced them into NBS cells. As shown in Figure 4E, consistent with our observation that acetylation of

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22 NBS1 inhibits phosphorylation of NBS1, mu tation of lysine to glutamine in NBS1 resulted in a significant decrease in N BS1 Ser343 phosphorylatio n. Importantly, the decrease in NBS1 phosphorylation is strictly proportional to the number of lysine mutations (i.e., the more lysine to glutamine change, the more decrease in NBS1 phosphorylation). Presence of SIRT1 Affects NBS1 phosphorylation Because SIRT1 is the chief r egulator of the acetylation st atus of NBS1 (Figure 3), we determined whether NBS1 Ser343 phosphor ylation was directly affected by the presence of SIRT1. As shown in Figure 5A, Nbs1 Ser343 was hypophosphorylated in Sirt1 -/MEFs compared to wild-type MEFs. Likewise, depletion of SIRT1 by siRNA resulted in a significant decrease in NBS1 Ser343 phosphorylation (Figure 5B). Acetylation of NBS1 Affects RDS Although results from one study sugges t that ATM phosphorylation of NBS1 contributes to the formation of IR-induced foci in cells (Zhao et al., 2000), other studies convincingly demonstrated that NBS1 phosphorylation does not affect formation of IR-induced foci and that the MRN complex is recruited to foci independently of ATM (Lim et al., 2000). We examined the ab ility of the NBS1 acetylation-defective mutant to associ ate with MRE11 and RAD50 and found that no significant differences exist between mu tant and wild-type NBS1 (Figure S1). Furthermore, no significant differences in NBS1 foci formation could be detected

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A Sirt1 + / + + + + B 23 Figure 5. Presence of SIRT1 affects NBS1 Ser343 phosphorylation (A) Murine Sirt1 +/+ and Sirt1 -/fibroblasts were irradiated (6 Gy) or left unirradiated. At different time points after IR, cell lysates were collected and analyzed by Western blotting with anti-pS343-mNbs1 antibodies. The blot was sequentially stripped and re-probed with anti-mNbs1 and pS1981-ATM antibodies to confirm equal mNbs1 protein levels and intact ATM activation. (B) 293T cells infected with adenoviruses expressing SIRT1 siRNA or control viruses were irradiated (6 Gy) or left unirradiated. At different time points after IR, cell lysates were collected and analyzed by Western blotting with anti-pS343-NBS1 antibodies. The blot was stripped and re-probed with anti-NBS1. between Sirt1 +/+ and Sirt1 -/MEFs following IR treatment (data not shown). Therefore, our data suggest that, similar to phosphorylation, acetylation of NBS1 does not play a role in the recruitment of MRN to H2AX domains surrounding DNA DSBs. Unlike IR-induced foci formation, phosphorylation of NBS1 is clearly involved in regulating the intra-S-phase checkpoint (Lim et al., 2000; Zhao et al., 2000). A common phenotype of NBS cells is the loss of the IR-induced S-phase checkpoint, leading to RDS. Using an RDS assay, we examined the effects of NBS1 acetylation on cell cycle checkpoints activated by DNA damage. NBS cells expressing Myc-NBS1 and enhanced green fluorescent protein (EGFP) were irradiated or mock-treated immediately after 25-bromodeoxyuridine (BrdU) treatment, and the ratios of BrdU-positive/EGFP-positive cells to EGFP-positive Post IR (min) Sirt1 / 0 30 60 0 + 30 + 60 + IB anti-pS343-Nbs1 pS343-Nbs1 IB anti-Nbs1 Nbs1 IB anti-pS1981-ATM pS1981-ATM IB anti-pS343-NBS1 IB anti-NBS1 Post IR (min) 0 30 60 0 30 60 + + + SIRT1 siRNA Control siRNA + + +

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24 cells were determined. Compared to wild-type NBS1, PCAF-hyperacetylated NBS1 was ineffective at inhibiting RDS, suggesting that hypoacetylation of NBS1 is crucial for intra-S-phase checkpoint ac tivation (Figure 6A). In contrast, PCAF did not alter the ability of the acetylation-re fractory mutant NBS1 to inhibit RDS. Similarly, in control experiments PCAF had no effect on the NBS1 phosphorylation mutant (S343A) in inhibi ting RDS. Western blot analysis confirmed that Ser343 phosphorylation of wildtype (Myc-NBS1), but not the acetylation mutant (Myc-NBS1mt), decr eased with increased acetylation in NBS cells. To be certain that NBS1 acetylat ion/deacetylation affects RDS, we transfected NBS cells (GM07166) with plas mids expressing wild-type or mutated NBS1, in the presence or absence of pl asmids that express PCAF. Stable clones were selected by growth in G418 plus or minus puromycin, and cells stably expressing NBS1 and/or PCAF we re treated with IR and examined for DNA synthesis using radiolabeled thym idine. Consistent with transient transfection results, acetylation of wild-t ype, but not acetylation-defective, NBS1 by PCAF significantly reduced its ability to inhibit RDS (Figure 6B). Finally, consistent with observations t hat NBS1 hyperacetylation prevents its ability to inhibit RDS in transfected NBS cells, we found that Sirt1 -/MEFs, which contain hypophosphorylated and hyperacetylated Nbs1, display a higher level of RDS compared to Sirt1 +/+ cells (Figure 6C).

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25 A 120 48 0 0 BrdU +EGFP +/EGFP +cell (+IR) BrdU + EGFP + /EGFP + cell (-IR) Myc-NBS1mt + + Myc-NBS1 + + + Flag-PCAF + + + Myc-NBS1(S343A) + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-Flag IB anti-pS343-NBS1 Flag-PCAF + + + + + + + + IR (15 Gy) + + + + + + + + Myc-NBS1 + + + + + Myc-NBS1mt + + + + + + + Myc-NBS1(S343A) 25 50 75 DNA synthesis (% 100 Myc-NBS1mt + + Myc-NBS1 + + + Flag-PCAF + + + Myc-NBS1(S343A) + + B IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-Flag IB anti-pS343-NBS1 Flag-PCAF + + + + + + + + IR (15 Gy) + + + + + + + + Myc-NBS1 + + + + + Myc-NBS1mt + + + + + + + Myc-NBS1(S343A) 25 50 75 DNA synthesis (% + Sirt1+/+ + Sirt1-/100 C

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26 Figure 6. NBS1 Acetylation Affects Radioresistant DNA synthesis (RDS) (A) NBS cells were transfected with plasmids encoding the indicated proteins. Twen ty-four hours after transfection, cells were irradiated (15 Gy) or left unirradiated. Left panel RDS was determined by comparing the ratio of BrdU + /EGFP + cells to EGFP + cells. The bar represents the ratio in the IR-treated cells divided by the ratio in the untreated cells. Right panels, to assess immunoprecipitation efficiency, protein expr ession, and NBS1 acetylation/phosphorylation, cell lysates were subjected to Western blot analysis or to high stringency immunoprecipitation/We stern blot analysis, as indicated. Representative blots are shown. (B) Left panel, NBS cells (GM 07166) transfected with vector alone or the indicated expression plasmids were grown in the presence of G418 (200 mg/ml, for cells that received NBS1 expression plasmids) and puromycin (0.5 mg/ml, for cells that received PCAF expre ssion plasmids) for 10 days. St able colonies were isolated, pooled together, and after treatment with 15 Gy of IR or left untreated, cells were radiolabeled with thymidine for RDS analysis. Right panels, representative blots to monitor immunoprecipitation efficiency, protein expression, and NBS1 acetylation/phosphorylation. (C) 2 X 10 4 Sirt1 +/+ or Sirt1 -/MEFs were plated on 35 mm di shes for 48 h. RDS assays were performed using the radio-labeling method to deter mine inhibition of DNA synthesis after 15 Gy of IR. Acetylation of NBS1 Affects Cell Survival Unsuccessful DNA damage responses are refl ected in an overall decrease in cell survival. Therefore, clonogenic cell surv ival assays were employed to examine the importance of NBS1 acetylation in DNA damage responses. NBS cells were exposed to a sublethal dose of IR, and col onies were counted two weeks later. Cell transfection and imm unoprecipitation efficiency, protein expression levels, and NBS1/NBS1mt acetylation levels we re monitored by immunoprecipitation followed with Western blots or by direct Western blots (Figure 7A). Consistent with the finding that hy peracetylated NBS1 negatively affects cell cycle checkpoint activation, PCAF-hyperacetyl ated NBS1 was much less effective in reducing the IR sensitivity of NBS cells (increasing cell survival) than nonhyperacetylated NBS1. As expected, in the presence of PCAF, survival among

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27 Figure 7. NBS1 Acetylation Affects Cell Survival Left panel, NBS cells transfected with plasmids (4 ug each) encoding the indicated proteins were irradiated (2 Gy) or left unirradiated. The surviving colonies were counted two weeks later. Right panels, direct Western blots and high A 20 40 60 % Survi 80 Myc-NBS1mt + + Myc-NBS1 + + + Flag-PCAF + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-Flag IR (2 Gy) + + + + + + Flag-PCAF + + + + + + + Myc-NBS1mt Myc-NBS1 + + + + + + + 20 40 60 % Survi 80 Myc-NBS1mt + + + + Myc-NBS1 + + + + + Flag-PCAF + + + + + 2 Gy + + + + + + + + + + + + 5 Gy B IR (5 Gy) + + + + + + Flag-PCAF + + + + + + + Myc-NBS1mt Myc-NBS1 + + + + + + + IP anti-Myc IB anti-AcK IP anti-Myc IB anti-Myc IB anti-pS343-NBS1 IB anti-Flag C 80 60 20 40 % Survi Sirt1+/+ + + Sirt1-/+ + IR (2 Gy) + + IR (5 Gy) + +

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28 stringency immunoprecipitation/Western blot analyses were performed to determine NBS1 acetylation, immunoprecipitation efficiency, and protein expressi on levels. Representative blots are shown. (C) Sirt1 +/+ and Sirt1 -/MEFs were irradiated with the indicated dosage or left unirradiated. The surviving colonies were counted two weeks later. Fractional cell survival is the fraction of colonies surviving irradiation divided by the tota l number of colonies in the unirradiated parallel culture. The data are expressed as mean SD from three separate experiments. cells expressing mutant NBS1, which is le ss efficiently acetylated by PCAF, was higher compared to cells expressing wild-type NBS1. To confirm that hyperacetylation of NBS1 negatively affects cell survival, we repeated the colony survival assays in cells stably expressing wild-type or mutant NBS1 and PCAF. As shown in Fi gure 7B, the presence of PCAF reduced the ability of wild-type NBS1 to increase ce ll survival after IR treatment in stably transfected NBS cells. Likewise, in complementary experiment s, the absence of Sirt1 decreased survival rate following -IR compared to wild-type MEFs (Figure 7C). Discussion HATs and HDACs play critical roles in di verse biological functions. In humans, two well-characterized HATs, hGCN5 and TIP60, play important roles in DNA repair (Brand et al., 2001; Ikura et al., 2000). In the current stud y, we show that NBS1 can be acetylated by PCAF and p300 and that acetylation of NBS1 inhibits Ser343 phosphorylation. At this ti me, the exact mechanism by which acetylation/deacetylation of NBS1 modul ates phosphorylation is unclear. One plausible model is that hypoacetylat ion of lysines on NBS1 induces an intramolecular structural change in NBS1 that favors phosphorylation. Our data

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29 clearly indicate that NBS1 ac etylation results in a decreas e in cell survival and an increase in RDS most likely due to decreased NBS1 phosphorylation. Mass spectrometry analysis of NBS1 i ndicated that ten of the 70 lysine residues in NBS1 were acetylated. T he NBS1 protein possesses three lysine acetylation consensus motifs, Kx 1-2 x/KK, (previously descr ibed by Yang, 2004), at residues 233-237 (KGHKK), 683687 (KNFKK), and 686-690 (KKFKK). However, we found that only two out of the ten potential acetylation sites identified by sequence analysis were actu ally acetylated (residues 233 and 690). Moreover, eight other acet ylation sites detected on NBS1 by mass spectrometry analysis do not conform to any consensuses. Therefore, not only does substrate recognition by SIRT1 not dependent on the amino acid sequence proximate to the acetylated lysine (Blander et al ., 2005), it appears that the NBS1 acetyltransferase(s) similarly does not have strict sequence requirements. Although we cannot rule out the possi bility that in addition to p300 and PCAF more acetyltransferases can modi fy NBS1, our data clearly demonstrate that NBS1 is deacetylated pr edominantly, if not exclus ively, by SIRT1. Our results are consistent with a model in which SIRT1 is required to maintain hypoacetylated NBS1, which promot es efficient IR-induced NBS1 phosphorylation and the ensuing cellular responses to DNA damage. Thus, an additional function of SIRT1 may be to maintain NBS1 in a constant hypoacetylated state. Alter natively, IR may induce SIRT1 activity in order to regulate the acetylation st atus of NBS1; however, th is possibility cannot be tested directly due to the low basal leve ls of acetylated NBS1. Despite this

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30 dilemma, these results are in accordance with the notion that SIRT1, in addition to silencing transcription, plays an im portant role in suppressing genomic instability and, consequently, in regulating aging in ma mmals (Lombard et al., 2005). Many evidences suggest a close lin k between histone deacetylation and DNA repair. For example, the yeas t Sin3/Rpd3 complex has been shown to modulate DNA DSB repair through the deacet ylation of histone H4K16 (Jazayeri et al., 2004). The Sir2 and Rpd3 proteins are recruited to HO lesions during homology-directed repair (T amburini and Tyler, 2005). RPD3 and HOS2 are required for the activation of DNA damage-inducible genes RNR3 and HUG1 (Sharma et al., 2007). Furthermore, HDAC4 is recruited to DSBs in vivo and, therefore, is implicated in DNA repair in mammalian cells (Kao et al., 2003). Knockdown of HDAC4 expression by RNAi increases radiosensitivity and blocks G2/M checkpoint maintenance. Also, it was reported that the SMRT-HDAC3 complex is involved in cellular recovery from DNA DSBs in mammalian cells (Yu et al., 2006). SMRT and HDAC3 are required for transcriptional repression by Ku70, a subunit of the DNA-PK DSB repair complex. Although p53 is known to be a substrate for SIRT1 (Luo et al., 2001; Vaziri et al., 2001), the contri bution of SIRT1 to p53-m ediated DNA damage repair is controversial. While one study suggests that SIRT1 modulat es p53-dependent DNA-damage responses (Chen et al., 2005) other reports argue that SIRT1 does not affect p53-mediated biological activities (Kamel et al., 2006; Solomon et al., 2006). More recently, SIRT6 was f ound to be a chromatin-associated protein

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31 that promotes normal base ex cision repair but does not play a role in regulating cell cycle checkpoints (Mostoslavsky et al., 2006). Our finding that SIRT1 deacetylates NBS1 and is intimately in volved in the regulation of the intra-Sphase checkpoint, coupled with the recent report that DNA damage induces SIRT1 expression (Wang et al., 2006), raises the intriguing possibility that HDACs ensure genomic stability through a variety of mechanisms involving multiple pathways. In the MRN complex, besides acetylation/deacetylation and phosphorylation of NBS1, the activities and functions of MRE11 are regulated by posttranslational modifications. MRE11 undergoes cell cycle-dependent phosphorylation in response to several ge notoxic agents, and this modification requires NBS1 but not ATM (Dong et al., 1999; Yuan et al., 2002). In addition, MRE11 is methylated at arginine residues by PRMT1, and mutation of the arginines severely impairs the exonucle ase activity of MRE11 although the mutated protein still forms the MRN comple x (Boisvert et al., 2005). In sharp contrast to the hypoacetylat ion of NBS1, cells containing hypomethylated MRE11 display intra-S-phase DNA damage checkpoint defects. In preliminary studies, we found that MRE11, but not RAD50, is acetylated in the cell (data not shown). Because SIRT1 associates with the entir e MRN complex, future studies are warranted to determine whether MRE11, lik e NBS1, is similarly regulated by SIRT1 and if so, whether acetylation/deacetylation of MRE11 interacts with other MRE11 modifications to ultimate ly affect the DNA DSB repair pathway.

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32 References 1. Aizawa, H., Hu, S.C., Bobb, K., Ba lakrishnan, K., Ince G., Gurevich, I., Cowan, M., and Ghosh, A. (2004). Dendrite development regulated by CREST, a calcium-regulated transcr iptional activator. Science 303 197-202. 2. Bakkenist, C.J., and Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylatio n and dimer dissociation. Nature 421 499-506. 3. Blander, G., and Guar ente, L. (2004). The Sir2 fam ily of protein deacetylases. Annu. Rev. Biochem. 73 417-435. 4. Blander, G., Olejnik, J., Krzymanska-Olejnik, E., McDonagh, T., Haigis, M., Yaffe, M.B., and Guarente, L. (2005). SIRT1 shows no s ubstrate specificity in vitro. J. Biol. Chem. 280 9780-9785. 5. Boisvert, F. M., Dery, U., Masson, J.Y., and Richard, S. (2005). Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control. Genes Dev. 19 671-676. 6. Brand, M., Moggs, J.G., Oulad-Abdelghani, M., Lejeune, F., Dilworth, F.J., Stevenin, J., Almouzni, G., and Tora, L. (2001). UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation. EMBO J. 20 3187-3196. 7. Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., Mostoslavsky, R., Cohen, H.Y. et al (2004). Stressdependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303 2011-2015.

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36 33. Liszt, G., Ford, E., Kurtev, M., and Guarente, L. (2005). Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J. Biol. Chem. 280 21313-21320. 34. Lombard, D.B., Chua, K. F., Mostoslavsky, R., Franco, S., Gostissa, M., and Alt, F.W. (2005). DNA repair, genome stability, and aging. Cell 120 497-512. 35. Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., and Gu, W. (2001). Negative control of p53 by Sir 2alpha promotes cell survival under stress. Cell 107 137-148. 36. Marks, P.A., Miller, T., and Richon, V.M. (2003). Histone deacetylases. Curr. Opin. Pharmacol. 3 344-351. 37. Maser, R.S., Zinkel, R., and Petrini, J.H. (2001). An alternative mode of translation permits production of a vari ant NBS1 protein from the common Nijmegen breakage syndrome allele. Nat. Genet. 27 417-421. 38. Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., and Horikawa, I. (2005). Evolutionarily conserved and nonc onserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 16 4623-4635. 39. Mirzoeva, O.K., and Petrini, J. H. (2001). DNA dam age-dependent nuclear dynamics of the Mre11 comp lex. Mol. Cell. Biol. 21, 281-288. 40. Mostoslavsky, R., Chua, K.F., Lombard, D.B., Pang, W.W., Fischer, M.R., Gellon, L., Liu, P., Mostoslavsky, G., Franco, S., Murphy, M.M., et al (2006). Genomic instability and aging-like phenotype in the absence of mammalian SIRT6. Cell 124 315-329.

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37 41. Nemoto, S., Fergusson, M.M., and Fi nkel, T. (2005). SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1a. J. Biol. Chem. 280 16456-16460. 42. North, B.J., Marshall, B.L., Borra, M.T., Denu, J.M., and Verdin, E. (2003). The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11 437-444. 43. Peterson, C.L. (2002). HDAC's at work : everyone doing their part. Mol. Cell 9 921-922. 44. Rodgers, J.T., Lerin, C., Haas, W., Gygi, S.P., Spiegelman, B.M., and Puigserver, P. (2005). Nutrient contro l of glucose homeostasis through a complex of PGC-1alph a and SIRT1. Nature 434 113-118. 45. Roth, S.Y., Denu, J.M., Allis, C.D. (2001) Histone acetyltransferases. Annu. Rev. Biochem. 70, 81-120. 46. Ruijter, A.J.M., van Gennip, A.H., Ca ron, H.N., Kemp, S., and van Kuilenburg, A.B.P. (2003). Histone deacetylases (HDACs): characterization of the classical HDAC family. 370 737-749. 47. Shiloh, Y. (1997). Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Annu. Rev. Genet. 31 635-662. 48. Solomon, J.M., Pasupulet i, R., Xu, L., Mc Donagh, T., Curtis, R., DiStefano, P.S., and Huber, L.J. (2006). Inhibition of SIRT1 cataly tic activity increases p53 acetylation but does not alter cell survival following DNA damage. Mol. Cell. Biol. 26 28-38.

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38 49. Sterner, D.E., and Be rger, S.L. (2000). Acet ylation of histones and transcription-related factors. Microbiol. Mol. Biol. R. 64, 435-459. 50. Sui, G., Soohoo, C., Affar el, B., Gay, F., Shi, Y., Fo rrester, W.C., and Shi, Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99 5515-5520. 51. Tamburini, B.A., and Ty ler, J.K. (2005). Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of doublestrand DNA repair. Mol. Cell. Biol. 25 4903-4913. 52. Tauchi, H., Kobayashi, J., Morishima, K., van Gent, D.C., Shiraishi, T., Verkaik, N.S., vanHeems, D., Ito, E., Nakamura, A., Sonoda, E. et al (2002). Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420 93-98. 53. Tauchi, H., Matsuura, S., Kobayashi J., Sakamoto, S ., and Komatsu, K. (2002). Nijmegen Breakage Syndrome gene, NBS1, and molecular links to factors for genome stability. Oncogene 21, 8967-8980. 54. van der Burgt, I., Chrzanowska, K.H ., Smeets, D., and Weemaes, C. (1996). Nijmegen breakage syndrome. J. Med. Genet. 33, 153-156. 55. Vaquero, A., Scher, M., Lee, D., Erdjument-Brom age, H., Tempst, P., and Reinberg, D. (2004). Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin. Mol. Cell 16 93-105. 56. Vaquero, A., Scher, M.B., Lee, D.H., Sutton, A., Cheng, H.L., Alt, F.W., Serrano, L., Sternglanz, R., and Reinber g, D. (2006). SirT2 is a histone

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39 deacetylase with preference for histone H4 Lys 16 during mitosis. Genes Dev. 20, 1256-1261. 57. Varon, R., Vissi nga, C., Platzer, M., Cerosaletti, K.M., Saar, K., Beckmann, G., Seemanova, E., Cooper, P.R., Nowak, N.J., Stumm, M., et al. (1998). Nibrin, a novel DNA double-strand brea k repair protein, is mutated in Nijmegen breakage syndrome. Cell 93, 467-476. 58. Vaziri, H., Dessain, S.K ., Ng Eaton, E., Imai, S.I., Frye, R.A., Pandita, T.K., Guarente, L., and Weinberg, R. A. (2001). hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107 149-159. 59. Wang, C., Chen, L., Hou, X., Li, Z., Kabra, N., Ma, Y., Nemoto, S., Finkel, T., Gu, W., Cress, W.D., and Chen, J. (2006). Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage. Nat. Cell Biol. 8 10251031. 60. Wolffe, A.P. (1996). Hist one deacetylase: a regulator of transcription. Science 272 371-372. 61. Wu, X., Ranganathan, V ., Weisman, D.S., Heine, W.F., Ciccone, D.N., O'Neill, T.B., Crick, K.E., Pi erce, K.A., Lane, W.S., Rathbun, G. et al (2000). ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405 477-482. 62. Yang, X.J. (2004). Lysine acetylation and t he bromodomain: a new partnership for signaling. Bioessays 26 1076-1087.

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40 63. Yang, X.J., Ogryzko, V.V., Nishikawa J., Howard, B.H., and Nakatani, Y. (1996). A p300/CBP-associat ed factor that comp etes with the adenoviral oncoprotein E1A. Nature 382 319-324. 64. Yang, X.J., and Seto, E. (2003). Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression. Curr. Opin. Genet. Dev. 13, 143-153. 65. You, Z., Chahwan, C., Bailis, J., Hunter, T., and Russell, P. (2005). ATM activation and its recruitment to dam aged DNA require binding to the C terminus of Nbs1. Mol. Cell. Biol. 25 5363-5379. 66. Yu, J., Palmer, C., Alenghat, T., Li, Y ., Kao, G., and Lazar, M.A. (2006). The corepressor silencing mediator for retinoid and thyroid hormone receptor facilitates cellular recovery from DNA double-strand breaks. Cancer Res. 66, 9316-9322. 67. Yuan, S.S., Su, J.H., Hou, M.F., Y ang, F.W., Zhao,S., and Lee, E.Y. (2002). Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells. DNA Repair 1 137-142. 68. Zhang, Q., Yao, H., Vo, N., and Good man, R.H. (2000). Acetylation of adenovirus E1A regulates binding of t he transcriptional corepressor CtBP. Proc. Natl. Acad. Sci. USA 97 14323-14328. 69. Zhang, X., Wharton, W., Yuan, Z., Tsai, S.C., Olashaw, N., and Seto, E. (2004). Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichosta tin A and repression by HDAC3. Mol. Cell. Biol. 24 5106-5118.

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41 70. Zhao, S., Renthal, W., and Lee, E.Y. (2002). Functional analysis of FHA and BRCT domains of NBS1 in chro matin association and DNA damage responses. Nucleic Acids Res. 30 4815-4822. 71. Zhao, S., Weng, Y.C., Yuan, S.S., Lin, Y.T., Hsu, H.C., Lin, S.C., Gerbino, E., Song, M.H., Zdzienicka, M.Z., Gatti, R.A. et al (2000). Functional link between ataxia-telangiectasi a and Nijmegen breakage syndrome gene products. Nature 405 473-477.

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42 SECTION II HISTONE DEACETYLASE 9 MODULATES CELL SURVIVAL BY REGULATING THE ATDC-P53 COMPLEX Abstract HDAC9 is a class II HDAC that acts as a signal-responsive suppressor of cardiac hypertrophy. Several proteins, most not ably the myocyte enhancer-binding factor 2 (MEF2), have been found to partner with HDAC9. Through its interactions with MEF2, HDAC9 represses MEF2 acti vity. By using immunoaffinity chromatography, we have identified a novel HDAC9-interacting protein, ATDC, the product of the ataxia-telangiectas ia group D complementing gene. The ATDC gene was originally isolated on the basis of its ability to complement the ionizing radiation sensitivity defect of AT group D fibroblasts. We found that ATDC is an HDAC9 substrate and its biologi cal functions are finely controlled by HDAC9. ATDC sequesters p53 in the cytopl asm, facilitating cell survival by antagonizing the p53-mediated apoptotic pathway following ionizing radiation. By modulating the acetylation of AT DC, HDAC9 prevents ATDC-p53 complex formation, promoting IR-induc ed cell death. Importan tly, we found that ATDC acetylation may also play important ro les in regulating pancreatic and cervical carcinoma radiosensitivity and surviv al. Thus, our results have important

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43 implications regarding clinical treatment for radio-resistant tumors caused by amplified expression of ATDC. Introduction Histone deacetylases (HDACs ) are a family of enzymes whose functions have been overwhelmingly associated with gen e expression and chromatin dynamics (Hassig and Schreiber, 1997; Kuo and Al lis, 1998; Johnson and Turner, 1999; Peterson, 2002). 18 human histone deacetylases that share homology through their deacetylase domains have been characterized, and they may be broadly divided into three classes on the basis of homology to the yeast HDACs RPD3 (class I), HDA1 (class II) and Sir2 (class II I) (Gray and Ekstro m, 2001; Yang and Seto, 2003; Ruijter et al., 2003; Gregoretti et al., 2004; Marks et al., 2003). Class I HDACs consist of nuclear proteins that are generally small in size (40-55 kDa) and are ubiquitously expressed. In contrast, Class II HDACs are larger (ranging from 100 to 160 kDa), with expression patterns that tend to be tissue-specific (Ruijter et al., 2003; Ma rks et al., 2003). Recent studies have implicated the importance of HDACs in normal devel opment and abnormal HDAC expression to have a function in a number of human di seases (Johnstone et al, 2003; Kramer et al., 2001). The gene encoding the HDAC9 protein is located on chromosome 7p21 (Mahlknecht et al., 2002). The human HDAC9 open reading frame is 3036 bp long and encodes a 1011 a.a. protein with a predicted mo lecular weight of 111.3 kDa and an isoelectric point of 6.41 (Zhou et al., 2001). The HDAC9 gene

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44 encodes multiple protein isoforms, some of which display distinct cellular localization patterns (Petrie et al ., 2003), and HDAC9 could shuttle between cytoplasm and nucleus (Zhou et al., 2001; Zhang et al., 2002). HDAC9 contains a conserved deacetylase domain, represses reporter activity when recruited to a promoter, and utilizes histones H3 and H4 as substrates in vitro and in vivo (Zhou et al., 2001; Petrie et al., 2003). HDAC9 is expressed in a tissue-specific pattern, most highly in heart tissue (Zhou et al., 2001; Zhang et al., 2002; Petrie et al., 2003). It was report ed that HDAC9 could restri ct cardiac growth in response to stress (Zhang et al., 2002). The ATDC (ataxia telangiectasia group D complementing) gene was originally identified by its ability to complement t he radiosensitivity defect of a cell line (AT5BIVA) from the D comp lementation group of the autosomal recessive human genetic disease ataxia telangiectasia (A T) (Kapp et al., 1992; Leonhardt et al., 1994). There are multiple zinc finger mo tifs and an adjacent leucine zipper motif in ATDC (Leonhardt et al., 1994). Endog enous and overexpressed ATDC protein locate in the cytoplasm (Laderoute et al., 1996; Brzoska et al., 1995; Reymond et al., 2001). ATDC gene is significantly amp lified in human pancreatic and cervical carcinomas (Iacobuzio-Donahue et al., 2002; Zhang et al. 2005) Although ATDC could complement the radi osensitivity of cells, the underlying mechanisms and its role in tumorigenesis are largely unknown.

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45 In the present study, we f ound that ATDC is an acetylat ed protein which is finely controlled by HDAC9. As a cytoplasmi c protein, ATDC might facilitate cell survival by sequestering p53 in the cytoplasm and antagonizing the p53mediated apoptotic pathway following ionizing radiat ion. By modulating the acetylation of ATDC, HDA C9 could regulate the ATDC-p53 complex formation, promoting IR-induced cell d eath. Importantly, ATDC ac etylation may also play important roles in regulati ng pancreatic and cervical carcinoma radiosensitivity and survival. Materials and Methods Plasmids, Antibodies, and Viruses The following expression plasmids us ed in these experiments have been described previously: Flag-HDAC9 (Zhou et al., 2001), Flag-HDAC9 isoforms (Petrie et al., 2003), Flag-PCAF (Yang et al., 1996), HA-p300 (Aizawa et al., 2004), pRc/RSV-HA-CBP (Zhang et al., 2000), pC53-SN3 and BP-100 GL2 (Peng et al., 2003), GST-p53 and its delet ion mutants, pGL3 control (Promega). The plasmids encoding HA-ATDC and it s deletion mutants were generated by standard PCR and subcloning. HA-ATD C K102R and K116R were generated using the QuickChange Site-Directe d Mutagenesis kit following the manufacturers protocol (Stratagene). HDAC9 shRNA and control shRNA was purchased from openbiosystem. HDAC9 shRNA and control shRNA retroviruses were generated by following the manufactu rers protocol (openbiosystem).

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46 Mouse affinity purified monoclonal anti-Flag M2, rabbit affinity purified polyclonal anti-HA, were purchased from Sigma. Goat polyclonal anti-ATDC, goat polyclonal anti-p53 and m ouse monoclonal anti-p53 (c lone DO-1) antibodies were purchased from Santa Cruz Biot echnology. Rabbit polyclonal anti-acetyllysine was purchased from Upstate (Millipor e). Rabbit polyclonal anti-acetylatedLys382-p53 was purchased from Cell Signaling Technology. Cell Culture, Transfection, and Adenovirus Infection 293T, HeLa, SiHa were grown in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal calf seru m (FCS) and penicillin/streptomycin (pen/strep). The AT5BIVA cell line (GM0 5849) was obtained from Coriell Cell Repository and grown in minimum essential medium (MEM) with 10% FCS and pen/strep. All transfections were normalized with equal amounts of parental vector DNA. Transfections were performe d with Lipofectamine 2000 (Invitrogen) according to the manufacturers instructi ons. For viral infection, HeLa cells were infected with retrovirus for 24 h in DMEM by following the manufac turers protocol (openbiosystem). Purification of HDAC9 Containing Complex HeLa cells were infected with Adenovirus -HA-HDAC9-Flag or Adenovirus-GFP control. Affinity purification of HA and Flag-tagged HDAC9-containing complex was performed according to previously published method (Rezai-Zadeh et al., 2004). Pufified complexes were concent rated, resolved by SDS-PAGE, and

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47 analyzed by silver staining. A Coomassie blue-stained sample was prepared in parallel, and bands corresponding to HDAC9-associated proteins were excised and subjected to proteolysis with trypsin. Peptides from these mixtures were sequenced by microcapilary LC-MS/MS. Immunoprecipitation and Western Blot Analysis For immunoprecipitations, cells were lyse d in buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% NP-40, and protease inhibito r cocktail) containing either 500 mM NaCl (high stringency) or 150 mM NaCl (l ow stringency). The lysates were incubated with the primary antibody overnight at 4 o C. The resultant immunocomplexes were collected, was hed four times in lysis buffer, and resolved by SDS-PAGE. For immunoblo tting, samples were transferred onto nitrocellulose membranes. Membranes were probed with the appropriate antibodies. Proteins of interest we re visualized using the Chemiluminescent Detection Kit (Pierce). Ion Trap Mass Spectrometry 293T cells were transfected with the HA-ATDC expression plasmid and treated with TSA (1.3 M ) overnight. The cells were then lysed in high stringency buffer containing 10 mM NaB and 10 mM nicotinamide. Cell extracts were subjected to immunoprecipitation with ant i-HA antibodies. The immune complexes were resolved by SDS-PAGE and stained with colloidal Blue (Invitrogen). The HAATDC sequence was analyzed with an in-hous e algorithm (EnzOpt) for a dual

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48 enzyme strategy maximizing proteotypic peptide coverage of all lysines. The appropriate HA-ATDC gel band was excised and divided into two parts. Each part was subjected to in-gel reducti on and carboxyamidomethy lation followed by separate tryptic or chymotryptic digesti on. Acetylated peptides from each digest were detected and sequenced using microc apillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ linear quadrupole ion trap mass spectrometer. Data analysis was facilitated with SEQUEST and Proteomics Browser Suite (Thermo). GST Pull-Down Assay GST and GST-p53 deletion mutants were expressed and purified from bacteria using standard methods. Equimolar quantit ies of the various purified proteins were conjugated to glutathione-Sepharos e beads and incubated with 293T cell (transfected with Flag-ATDC) lysates for 1 h at 4C. After extensive washing, bound proteins were eluted and analyzed by Western blotting with anti-Flag antibodies. Colony Survival Assay Colony survival assays were performed as previously described (Zhao et al., 2002) with minor modifications. Briefly, 24 h after transfection, cells were plated in quadruplicate (1,000 cells per 60-mm ti ssue culture dish). The cells were irradiated with 0, 2, 2.5 or 7.5Gy as indicated. After two weeks, dishes were washed with PBS, fixed in ice-cold met hanol for 15 min, and then stained with

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49 Giemsa stain for 30 min. Colonies on each plate were quantif ied and expressed as the percentage of t he unirradiated control. Luciferase Assay Luciferase assay was performed as previ ously described (Zhang et al., 2004). Briefly, U2OS cells were plated in 6-well dishes at a density of 4 x 10 5 cells/well. 24hr after transfection, cells were treated as indicated in figures before harvesting with 250 l of passive lysis buffer (Promega). Protein concentrations of all samples were determined using Bradford reagent (Bio-R ad), and the relative light unit values were measured with firefly assay reagent (Promega) and a luminometer. Immunofluorescence Assay Cells were washed with PBS, fixed in 4% paraformaldehyde for 10min at room temperature. Cells were permeabilized for 15min at room temperature using 0.5% TritonX-100 in PBS. After blocking by 1% BSA in PBS for 30min, cells were incubated with primary antibody at cold room overnight, followed by Alexa-555 and Alexa-488-conjugated secondary antibody for 1h at room temperature. Wash three times with PBS, dry slides, and m ount slides with Bectashield mounting medium with DAPI. Pictures were tak en using Zeiss confocal microscopy.

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Cell Apoptotic Assay Cell apoptotic assay was performed as previously described (Sawada et al., 2003) with minor modifications. Briefly, apoptosis was induced by -irradiation (5Gy for AT5BIVA cells and 10Gy for U2OS cells). To examine the effects of ATDC or HDAC9, 10 cells were cotransfected with 0.1 ug of pEGFP-C3 (GFP expression plasmid, Clonetech) to mark the transfected cells. The vector plasmid of pcDNA3.1 (+) was used as a negative control. One day after transfection, cells were treated with -irradiation. Two days after treatment, cells were stained with DAPI and apoptotic nuclei were counted in GFP-expressing cells under a fluorescence microscope (three hundred cells were counted for each experiment). Each point in figures showing percentages of apoptosis represents the mean 5 s.e.m of three experiments. Results Immunoaffinity Purification of HDAC9 Containing Complex To further characterization of HDAC9 biological function, we used an epitope-tagging strategy to isolate HDAC9-containing protein complexes from human cells. We generated an adenovirus encoding double-tagged human HDAC9 protein containing N-terminal HA and C-terminal Flag epitopes (HA-HDAC9-Flag) (Figure 8A) using the pAdEasy-1 system (He et al., 1998). To isolate protein complexes containing HDAC9, HeLa cells were infected with Ad-HA-HDAC9-Flag or Adenovirus vector for 36hrs. Whole cellular extracts from the infected 50

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51 cells were sequentially subjected to affinity chromatography on M2 (Flag antibody) agarose beads and an HA-affinity co lumn. Finally, the bound proteins were fractionated by SDS-PAGE and visualized by silver staining (Figure 8B). A major protein band of 65 kDa was copurified with HDAC9 from HA-HDAC9-Flagexpressing cells (Figure 1B, lane 2 and 4) but not from virus control cells (Figure 8B, lane 1 and 3), suggesting that this prot ein is a specific binding partner of HDAC9. By peptide sequencing of the band usi ng mass spectrometric analysis, it was identified as a known protei n called ATDC (Accession # Q14134). ATDC Interacts with HDAC9 To confirm the interaction between HD AC9 and ATDC, we co transfected the Flag-HDAC9 and HA-ATDC plasmids transiently into 293T cells. Cell lysates were subsequently mixed with the antiHA agarose and the resulting immune complexes were collected and analyzed by immunoblotting with the anti-Flag antibody (Figure 8C). As indicated in Figure 8C, immunoprecipitation of HAATDC from lysates of these cotransfected cells result ed in coprecipitation of Flag-HDAC9. We also detected this intera ction reciprocally by using the anti-Flag antibody for immunoprecipitation and anti-HA antibody for probing of the blotted precipitate (Figure 8D). These observati ons provided the first indication that HDAC9 and ATDC can form physical complexes with one another in vivo. To investigate the interaction between endogenous HDA C9 and ATDC, cell extracts from HeLa cells were i mmunoprecipitated with ant i-ATDC or with the

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A C Adenovirus-HA-HDAC9-Flag Input + 52 HDAC9 HA FLAG Mock IP + HA IP + B (kDa) 250 150 100 75 50 37 GFP HDAC9 -FLAG F-HDAC9 + + + -HA HA-ATDC + + + HDAC9 Marker GFP F-HDAC9 IB anti-Flag HA-ATDC IB anti-HA HDAC9 Input + D Mock IP + Flag IP + F-HDAC9 + + + ATDC HA-ATDC + + + IB anti-HA HA-ATDC IB anti-Flag F-HDAC9 G F-HDAC9C (601-1011) + 1 2 3 4 5 F-HDAC9M (351-600) + F-HDAC9N (1-350) + Mock IP + E F-HDAC9 FL + IP anti-ATDC + IP anti Flag + + + + + HA-ATDC + + + + + HDAC9 IB anti-HDAC9 IB anti-HA HA-ATDC ATDC IB anti-ATDC 1 2 F Mock IP + IB anti-Flag IP anti-HDAC9 + IB anti-ATDC IB anti-HDAC9 ATDC HDAC9 Input (no IP) HA-ATDC 1 2 IB Anti-HA 1 2 3 4 5

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53 Figure 8. Immuno-affinity purification of HDAC9 containing complex (A) Scheme of the Adenovirus-HA-HDAC9-Flag. (B) Purification of the HDAC9-containing complexes. HA-FLAG-epitopetagged HDAC9 was purified from HeLa cells infect ed with adenovirus expressing HA-HDAC9-FLAG by immunoprecipitation with antibody specific for FLAG (lane 2), followed by antibody s pecific for HA (lane 4). As a control, mock purification was performed from HeLa cells infected with adenovirus expressing GFP alone (lanes 1 and 3). Specific HDAC9-interacting protein bands were peptide sequenced by mass spectrometry. (C, D) 293T cells were co-transfected with plasmids (4ug each) encoding the i ndicated HA and Flag fusion proteins. Anti-HA and anti-Flag immunoprecipitates obtained under low stringency conditions were analyzed by West ern blotting with the indi cated antibodies. (E, F) Endogenous ATDC or HDAC9 were immunoprecipitated from HeLa cell lysates under low stringency conditions with antiATDC or anti-HDAC9 polyclonal antibodies. Immune complexe s were analyzed by Western blotting with indicated antibodies. (G) 293T cells were co-transfected with HA-ATDC and Flag-HDAC9 or its deletion mutants. Anti-Flag immunoprecipitates obtained under low stri ngency conditions were analyzed by Wester n blotting with anti-HA antibody. control IgG. As expected, Western blot analysis revealed that HDAC9 was clearly detected in the immunoprec ipitations obtained with the anti-ATDC antiserum (Figure 8E) but not the control IgG (F igure 8E). Conversely, endogenous ATDC was readily immunoprecipitat ed with the HDAC9-specific antibody (Figure 8F), but not with a control IgG (Figure 8F). These data indicate that HDAC9 and ATDC could form a physical complex in vivo. To further investigate how ATDC interacts with HDAC9, we generated several HDAC9 deletion mutants. HA-A TDC and Flag-HDAC9 deletion mutants were cotransfected into 293T cells. Cell lysates were immunoprecipitated with anti-flag agarose. As shown in Figure 8 G, ATDC specifically bound to HDAC9 Cterminal domain (HDAC9 601-1011aa) (Figure 8G ), but not to Flag vector control, the N-terminal domain of HDAC9 (1350aa) and the central domain (HDAC9 351600). Thus, the above findings demonstrat e that ATDC interacts with mammalian HDAC9 deacetylase in vivo.

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54 ATDC is an Acetylated Protein Since HDAC9 could form physical co mplexes with ATDC, we sought to determine whether ATDC could serve as a substrate of HDAC9 enzyme. Firstly, we examined whether ATDC could be modifi ed by acetylation. 293T cells were transfected with the plasmid encoding HA-A TDC. 24hrs post transfection, cells were incubated with ethanol carrier or HDAC chemical inhibitors trichostatin (TSA) overnight. We then immunoprecipitated HA-ATDC fr om these cells and analyzed the immune complex by western blotting using an antibody previously shown to recognize acetylated lysines in viari ous proteins. As shown in figure 9A, treatment with TSA significantly enhances ATDC acetylation. Anti-acetyl-lysine Western blot analysis of ant i-ATDC immunoprecipitates revealed that TSA also could enhance the acetylation state of endogenous ATDC (Figure 9B). To map the ATDC lysine-acetylation sites induced by TSA, tandem mass spectrometry analysis was performed. We found that at least 9 lysine sites of ATDC was acetylated (Figure 9C). These data strongly sugested th at ATDC was an acetylated protein within cells. ATDC Acetylation is Regulated by HDAC9 It is well known that the histone acet yltransferases PCAF and p300 target nonhistone proteins for acetylation (Glozak et al., 2005; Kouzarides, 2000; Yang, 2004). So, we co-transfected 293T cells wi th vectors expressing HA-ATDC and Flag-PCAF or HA-P300. The cell lysates were then analyzed by immunoprecipitation using anti-HA agarose followed by western blotting using

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B IP ATDC + + TSA + A IP IgG + + HA-ATDC + + TSA + + IP anti-HA Ac-ATDC IB AcK IB anti-AcK Ac-HAATDC ATDC IB ATDC IP anti-HA HAATDC IB anti-HA 1 2 3 4 1 2 C 55 102 116 291 275 300 361 478 554 495 B-BOX Coiled-coil F-HDAC9 + E HA-p300 + + HA-ATDC + + IP anti-HA HA-p300 + Ac-HA-ATDC D IB anti-AcK F-PCAF + HA-ATDC + + + IP anti-HA IB anti-AcK IP antiHA IB anti-HA Ac-HA-ATDC IP anti-HA HA-ATDC IB anti-HA HA-ATDC Input IB anti-HA HA-p300 Input F-PCAF Flag IB Input IB anti-Flag Input F-HDAC9 HA IB HA-p300 1 2 1 2 3 Control shRNA + F HDAC9 shRNA + Control shRNA + IP ATDC + + HDAC9 shRNA + Ac-ATDC AcK IB HDAC9 IB HDAC9 IgG H ATDC Actin IB Actin ATDC IB IgG H 1 2 1 2

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56 Figure 9. ATDC is an acetylated protein which is regulated by HDAC9 (A) 293T cells were transfected with plasmi ds that expressed HA-ATDC (4ug). Twenty-four hours post-transfection, cells were left untreated or treated overnight with TSA (1.3uM). Cell lysates were subjected to immunoprecipitation/Western blotting. (B) HeLa cells were treated with TSA (1.3uM) or left untreated overnight. Cell lysates were then immunoprecipitated under high stringency conditions with anti-ATDC antibodies. Endogenous acetylated ATDC was analyzed by Western blotting with a mixture of polyclonal anti-acetyl-lysine antibodies. (C) HA-ATDC, which was expressed and purified from 293T cells, was digested with trypsin and subjected to ITMS. Positions of the unambiguously-identified acetylated lysine resi dues are shown. (D) 293T cells were co-transfected with equal amounts (4ug) of HA-ATDC one of the following expression pl asmids: Flag-PCAF, or HA-p300. Cell lysates were immunoprecipitated (IP) under high stringency conditions using anti-HA antibodies. Immunoprecipitat es were subjected to Western blot (IB) analysis using anti-acetyl-lysine (AcK) antibodies. The blot was stripped and re-probed with anti-HA antibodies to confirm equal immunoprec ipitation efficiency and loading. Fl ag-PCAF and HA-P300 expression were detected in the cell lysate. (E) 293T cells were co-transfected with plasmids that express HA-ATDC, HA-P300, and either a vector or a Flag-HDAC9. Acetylation of HA-ATDC and all prot ein levels were determined with direct Western blotting or immunoprecipitations followed by Western blo tting using the indicated antibodies. (F) HeLa cells were infected with either retrovirus that expresses contro l siRNA or retrovirus that expresses HDAC9 shRNA. Western blots were performed with the indicated antibodies to assess the acetylation of endogenous ATDC and ATDC immunoprecipitation efficiency. Bottom two panels, western blot to show that HDAC9 shRNA is functional. anti-acetyl lysine antibody. Acetylated ATDC was readily detected with coexpression of P300, but not with PCAF acetyltransfera se (Figure 9D). We found ATDC also could interact with p300 within cells (data not shown). To further address ATDC acetylati on was regulated by HDAC9, we transfected 293T cells with HA-ATDC, P300 and Flag vector or Flag-HDAC9. HA-ATDC was immunoprecipit ated and followed by antiacetyl lysine western blotting. As shown in figure 9E, a high le vel of acetylated ATDC was found in the cells cotransfected with P300 and HA-ATD C (Figure 9E, lane 1); however, p300mediated ATDC acetylation levels were si gnificantly decreased by expression of Flag-HDAC9 (Figure 9E, lane 2). Fu rthermore, we assessed the ATDC

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57 acetylation in HDAC9 knock-down cells by immunoprecipitating endogenous ATDC and immunoblotting with antibodies to acetylated lysine (Figure 9F). Acetylation of endogenous ATDC was enhanced in HDAC9 knock-down cells compared to control shRNA cells (Figure 9F), suggesting that endogenous HDAC9 does influence ATDC acetylation in vivo. As indicated in Figure 9F, endogenous HDAC9 protein levels were reduced by the sequence-specific HDAC9 shRNA but not by control shRNA (Figure 9F, bottom panel). Taken together, these data implicate a strong ATDC deacetylation activity of HDAC9 deacetylase. Acetylation of ATDC was barely detectable in the absence of HDAC9 inhibiotr TSA or ex ogenous acetyltransferase p300 (Figure 9A and 9D), possibly owing to the limiting am ounts of endogenous p300 acetytransferase activity, or ATDC preferentially fo rming a stable comple x with the HDAC9 deacetylase. HDAC9 has multiple isoforms (Petrie et al., 2003). To test whether ATDC has HDAC9 isoform preference binding cap ability, 293T cells were cotransfected with the plasmids encoding HA-ATDC and Flag-tagged all known HDAC9 isoforms (Petrie et al., 2003) Cell lysates were immuno precipitated with anti-flag agarose, and then analyzed with anti-HA ant ibody. As shown in figure 10A, ATDC preferentially bound to HDAC9-PNAS, and weakly bound to HDAC9 E7, but not the other HDAC9 isofroms. Co rrelated with the binding preference, deacetylation of ATDC by HDAC9 might be also highly isoform-specific since only HDAC9-PNAS shows str ong deacetylase activity to ATDC (Figure 10B, lane

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58 2), but not other HDAC9 isoforms we tested (HDAC9 E7 or HDAC9-JBC) (Figure 10B, lane 3 and 4). HDAC7 and HDAC9 were further s ubdivided into the class IIa HDAC based on their homology (Gray et al., 2001) As indicated in figure 10C, the ATDC-HDAC9 interaction might also be highly HDAC specific since HDAC7 did not co-precipitate with ATDC under identical conditions (Figure 10C). Furthermore, p300-acetylated ATDC was s pecifically deacetylated by HDAC9 (Figure 10D, lane 3), but not by HDAC7 (Figure 10D, lane 2). HDAC9 Negatively Regulat es ATDC Biological Function ATDC was cloned and coined by its ability to complement the radiosensitivity defect of the AT5BIVA fibrobl ast cell line. To observe the effect of HDAC9 on its function, AT5BIVA cells were transfect ed with vector alone, HA-ATDC alone, Flag-HDAC9 alone or HA-ATDC and Flag-HDA C9 expression constructs. As expected, cell colony survival assay suggested ATDC could complement the AT5BIVA cell radiosensitivity defect (F igure 11A, compare column 1 and 3). Interestingly, HDAC9 coexpression signi ficantly antagonize the ATDC function, with about 25.00% of the ce ll survival compared with about 47.00% of ATDC alone-complemented cells (Figure 11A, compare column 3 and 4). It has been reported that -irradiation could induce cell apoptosis to a greater extent in AT5BIVA cells than in co ntrol fibroblast cell lines (Zhang et al., 2001; Jung et al., 1998). Combining the fa ct that ATDC could provide AT5BIVA

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F-HDAC9E7 + B 59 Figure 10. ATDC acetylation is preferentially regulated by HDAC9 (A, C) 293T cells were co-transfected with plasmids (4ug each) encoding the indicated HA and Flag fusion proteins. Anti-HA and anti-Flag immunoprecipitates obtained under low stringency conditions were analyzed by Western blotting with the indicated antibodies. HDAC9 isoforms used in panel A: 1. Vector Control; 2. HDAC9-PNAS; 3. HDAC9-JBC; 4. HDAC9CD; 5. HDAC9E7; 6. HDAC9CDE7; 7. HDAC9E12; 8. HDAC9E15. HDAC9-PNAS represents the first reported-HDAC9 gene product on PNAS in 2001, and this is the one we used to purify the HDAC9 containing complex. HDAC9-JBC represents the longest HDAC9 isoform published on JBC in 2003. (B, D) 293T cells were co-transfected with equal amounts (4ug) of HA-ATDC the HA-p300 expression plasmid. Cell lysates were immunoprecipitated (IP) under high stringency conditions using anti-HA antibodies. Immunoprecipitates were subjected to Western blot (IB) analysis HA-ATDC + + + + + + + + 75KD HA-ATDC HA-ATDC HD9 isoforms IP FLAG IB HA INPUT IB HA IP FLAG IB FLAG 1 2 3 4 5 6 7 8 HDAC9 isoforms A F-HDAC9 JBC + HA-p300 + + + + F-HDAC9 PNAS + HA-ATDC + + + + IP HA Ac-HA-ATDC IB AcK IP HA HA-ATDC IB HA F-HDAC9 isoforms INPUT IB FLAG HA-p300 INPUT IB HA 1 2 3 4 C F-HDAC9 + D F-HDAC9 + F-HDAC7 + F-HDAC7 + HA-p300 + + + HA-ATDC + + + HA-ATDC + + + Flag IP HA IP HA-ATDC HA IB Ac-HA-ATDC AcK IB HA IP Flag IP Flag-HD9 HA-ATDC HA IB Flag IB Flag-HD7 Input Input HA IB Flag-HD9 HA-ATDC Flag IB Flag-HD7 1 2 3 Input HA IB HA-p300 1 2 3

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60 using anti-acetyl-lysine (AcK) antibodies. The blot was stripped and re-probed with anti-HA antibodies to confirm equal immunoprecipitation efficiency and l oading. HA-P300 and HDACs expression were detected in the cell lysate. cells a radioresistant phenotype (Kapp et al., 1992; Hosoi et al., 1994), its interesting to hypothesize that ATDC mi ght facilitate AT5BIVA cell survival by inhibiting -irradiation induced-cell death. To te st this idea, apoptosis assay was carried out in AT5BIVA cells by expressing vector alone, HA-ATDC alone, FlagHDAC9 alone or HA-ATDC an d Flag-HDAC9. As shown in figure 11B, ATDC significantly decreases the IR-induced AT5BIVA cell apoptosis (Figure 11B, compare column 1 and 3). However, introduction of HDAC9 could strongly reverse the ATDC anti-apoptotic function (F igure 11B, compare column 3 and 4). A parallel western blot s hows HDAC9 does not affect the HA-ATDC expression in cells (Figure 11A, right panel). ATDC Antagonizes p53-mediated A poptotic Pathway Activated by irradiation and Forms a Ph ysical Complex with p53 P53 has been shown to be important for -irradiation-induced apoptosis (Lee et al., 2001; Herzog et al., 1998; Chong et al ., 2000). Next, we examined the effect of ATDC on p53-mediated apoptotic pathway As indicated in figure 12A, ATDC transfected-U2OS (wild type p53+/+) cells were more resistant to -irradiation induced-cell death, with about 22.3% of the cells apoptosis while the vector control-transfected cells showed significantly suscept ible to apoptosis under the same conditions (Figure 12A, compare co lumn 1 and 2). Furthermore, the same

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61 A 0.05.010.015.020.00035.040.045.050.01234 F-HDAC9 + + HA-ATDC + + 25.30. HA-ATDC Input % Survival IB anti-HA Input F-HDAC9 IB anti-Flag Input Actin IB anti-Actin 1 2 3 4 HA-ATDC + + F-HDAC9 + + AT5BIVA 01020304050601234 B Apoptotic cell (%) HA-ATDC + + F-HDAC9 + + AT5BIVA

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62 Figure 11. HDAC9 negatively regulates ATDC biological function (A) Left panel, AT5BIVA cells transfected with plasmids (4ug each) encoding the indicated prot eins were irradiated (2Gy) or left unirradiated. The surviving colonies were counted two weeks later. Right panels, direct Western blots were performed to determine protein expression levels. Representative blots are shown. (B) AT5BIVA cells were transfected as indicated (see Methods). One day after tr ansfection, cells were irradiated (10G y). After a further 48h, the number of apoptotic cells was counted in GFP+ cells. experiments were performed in the human p 53 null cell line H1299. No significant effect was observed on H1299 cells (F igure 12A, compare column 3 and 4). These data suggested that ATDC could facili tate cell survival by antagonizing the p53-mediated apoptotic pathway activated by -irradiation. To further explore the molecular me chanism by which ATDC affects p53 function, we tested whether ATDC intera cts with p53 in vivo. 293T cells (wild type p53+/+) were transfected with HA-ATDC plas mid. Cell lysates were subsequently mixed with the anti-HA agarose and t he resulting immune complexes were collected and analyzed by immunoblotting with the p53-specific monoclonal antibody (DO-1) (Figure 12B). As shown in figure 12B, immunoprecipitation of HA-ATDC from lysates of t hese transfected cells result ed in coprecipitation of endogenous p53. However, -irradiation doesnt affect the ATDC-p53 complex formation. These observations provided the first indication that ATDC and p53 can form physical complexes with one another in vivo. This association of ATDC and p53 also occurs in the absence of overexpression, epitope tagging of either protein in SiHa cells (Figure 12C), which express both endogenous ATDC and wild-type

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63 A 0510152025303540 Mock IP + + B HA IP + + Input + + IR + + + HA-ATDC + + + + + + p53 p53 IB Apoptotic cell (%) HA-ATDC HA IB 1 2 3 4 5 6 IP ATDC + C IP IgG + INPUT + IP ATDC p53 IB p53 1234HA-ATDC + + IP ATDC ATDC IB ATDC U2OS (p53+/+) H1299 (p53-/-) 1 2 3 D 1 2 3 4 5 HA IP p53 IB HA IP HA IB Vec FL 1-200 181-360 181-588 p53 B-BOX Coiled-coil p53 p53 p53 FL (Lane 2) 1-200 (Lane 3) 181-360 (Lane 4) 181-588 (Lane 5)

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64 Figure 12. ATDC facilitates cell survival by antagonizing p53-mediated apoptotic pathway activated by girradiation and ATDC forms a physical complex with p53 (A) U2OS and H1299 cells were transfected as indicated (see Methods). One day afte r transfection, cells were irradiated (10Gy). After a further 48h, the number of apoptotic cells was counted in GFP+ cells. (B) 293T cells were transfected with HA-ATDC. 24hrs post transfection, cells were irradiated (10G y) or left untreated. One hour after irradiation, HA-ATDC was immunoprecipitated under low stringe ncy conditions with anti-HA agarose. Immune complexes were analyzed by Western blotting with p53 antibody DO-1. (C) Endogenous ATDC was immunoprecipitated from SiHa cell lysates under low stringency conditions with goat anti-ATDC polyclonal antibodies. Immune complexes were analyzed by Western blotting with p53 antibody DO-1. (D) 293T cells were transfected with HA-ATDC or its deletion mutants. Anti-HA immunoprecipitates obtained under low stri ngency conditions were analyzed by West ern blotting with p53 antibody DO-1. p53 proteins. Analyses of three different HA-ATDC deletions indicated that p53 preferentially interacts with the N-termi nus of ATDC (residues 1-200) (Figure 12D). ATDC Inhibit p53 Function by Sequestering p53 into the Cytoplasm ATDC mainly located in the cytoplasm (L aderoute et al., 1996; Brzoska et al., 1995; Reymond et al., 2001). To further confirm the ATDC subcellular localization, U2OS cells were transfe cted with the plasmid encoding HA-ATDC, and immunofluorescence study was carried out. Consistent with the previous findings (Brzoska et al., 1995; Reymond et al., 2001), HA-ATDC was found in the cytoplasm in a ribbon-like structure (Figure 13A). Endogenous ATDC was also found in the cytoplasm in SiHa cells (Figure 13B) and pancreatic carcinoma tissues (Figure 13C). Since ATDC involv es in regulating p53-mediated apoptotic pathway, its intriguing to test whether ATDC directly controls subcellular localization of p53. To test this idea, U2OS cells were transfected with HA-ATDC.

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A HA-ATDC DAPI Merged B ATDC Merged DAPI C Figure 13. ATDC is a cytoplasmic protein (A) U2OS cells were transfected with HA-ATDC. 24hr post transfection, cells were fixed, permeabilized, and immunostained with anti-HA antibody. (B) SiHa cells were fixed, permeabilized, and immunostained with anti-ATDC antibody, followed by Alexa-555 secondary antibody. (C) Pancreatic carcinoma tissue samples were stained with anti-ATDC antibody. As shown by immunofluorescence staining with the p53-specific antibody DO-1 (Figure 14A, inset), p53 mainly located in the nucleus in the absence of overexpressed-ATDC. Strikingly however, after overexpression of ATDC, p53 65

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66 was predominantly relocalized to the cyt oplasm of these cells and displayed a similar pattern as ATDC (Figure 14A). Sim ilar results were also obtained in the cells treated with -irradiation (Figure 14A, bottom panel). These data indicated that forced-expression of ATDC could control p53 subcellula r localization even under stress. To test the effect of ATDC on endogenous p53 transcriptional activity, U2OS cells were transfected with the p53-responsive BP100-luciferase reporter and different dosage of plasmid encoding HA-ATDC. P53 transcription function was measured by luciferase assay. As shown in figure 14B, ATDC shows strong inhibition of endogenous p53 tran scriptional activity (Figure 14B) in a dose dependent manner. The inhibitory effect of ATDC on p53 responsive promoter is quite specific, since the pGL3 control reporter was not affected by ATDC. ATDC even shows similar strong inhibitory effect on p53 transcriptional activity under stress response (Figure 14C). HDAC9 Regulates the ATDC-p53 Complex Formation and Function Since ATDC is a HDAC9 substrate as an acetylated protein, it is interesting to address whether HDAC9 could regulate the ATDC-p53 complex formation. To test this idea, 293T cells were trans fected with vector, HA-ATDC alone or HAATDC and Flag-HDAC9. The cell lysates were immunprecipitated with anti-HA agarose and the resulting immune complexes were collected and analyzed by immunoblotting with the goat anti-p53 polyclonal antibody Consistent with the above results, ATDC could efficiently pull-down endogenous p53 (Figure 15A, lane 2). However, HDAC9 could significantly inhibit the ATDC and p53 interaction

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A IR 67 Figure 14. ATDC inhibits p53 function (A) U2OS cells were transfected with HA-ATDC. 24hr post transfection, cells were left untreated or treated with 10Gy -irradiationn. 1hr after treatment, cells were fixed, permeabilized, and stained with anti-HA and anti-p53 DO-1 antibody, followed by Alexa-555 and Alexa-488-conjugated secondary antibodies. Samples were analyzed using Zeiss Confocal HA-ATDC p53 Merged + HA-ATDC p53 Merged 0200000400000 Relative Luciferase Activity 6080101214 00000000000000000000000160000018000002000000123456 B 78 BP-100 GL2 + + + + + pGL3-Control + + + HA-ATDC (ug) 0 0.5 1 2 4 0 1 4 C 050000100000150000200000250000300000 350400450500550600650700Relative Luciferase Activit y 000000000000000000000000750000800000850000900000950000123456 BP-100 GL2 + + + + + + HA-ATDC (ug) 0 1 4 0 1 4 IR + + +

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68 Microscopy. (B) U2OS cells were transfected as indicated. Luc iferase activities were determined in triplicate. Error bars show standard deviations. (C) U2OS cells were transfected as indicated. 24hr post transfection, cells were left untreated or treated with 10Gy -irradiation. 4hr after treatment, cells were harve sted and luciferase activities were determined in triplicate. Error bars show standard deviations. (Figure 15A, compare lane 3 and 2). Furt her, treatment with HDAC inhibitor TSA could significantly enhance the ATDCp53 complex formation (Figure 15B, compare lane 2 and 3). These data suggest ed that acetylated form of ATDC preferentially interacts with p53 in cells. Correlated with the above results, T SA treatment could significantly increase the ATDC suppressive effect on endogenous p53 transcriptional activity (Figure 15C). Furthermore, expression of p21, a major transcriptional target of p53, was significantly induced by ATDC-specific siRNA (Figure 15D, left panel) or by overexpression of HDA C9 (Figure 15D, right panel ) in SiHa cells although the total level of p53 was unchanged. ATDC K116 Acetylation is the Ma jor Functional Acetylation Site Responsible for Regulating p53 Interaction Two acetylation sites of ATDC (K102 and K116) located in the p53 binding domain (ATDC 1-200aa) (Figur e 9C and 12D). By aligni ng the protein sequence among several known vertebrates AT DC homologous (human, chimpanzee, mouse, rat and dog) (Figure 16A), we f ound that lysine 102 was replaced by arginine in mouse and rat ATDC. Since arginine mutation could mimic the nonacetylated form of lysine (Chan et al., 2001), this suggests K102 acetylation

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TSA + B F-HDAC9 + A HA-ATDC + + HA-ATDC + + HA IP p53 p53 IB HA IP 69 p53 IB HA IP HA IB HA IP Flag IB p53 HA IP HA-ATDC HA IB HA-ATDC 1 2 3 F-HDAC9 1 2 3 Ad-GFP + Control SiRNA + D Ad-HA-HDAC9-Flag + ATDC SiRNA + C 0.01.02.03.0 4.5.6.7.8.Relative Luciferase Activity (x105 000009.010.0123456 IB anti-p21 IB anti-p21 p21 IB anti-p53 IB anti-p53 p53 IB anti-Flag IB anti-ATDC ATDC IB anti-actin IB anti-actin Actin 1 2 1 2 ) BP-100 GL2 + + + + + + HA-ATDC (ug) 0 0 1 1 4 4 Eth + + + TSA + + +

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70 Figure 15. HDAC9 regulates the ATDC-p53 complex (A) 293T cells were co-transfected with HA-ATDC and Flag-HD AC9 or control vector. Anti-HA immunoprecipitates obtained under low stringency conditions were analyzed by West ern blotting with p53 antibody DO-1 and anti-Flag. (B) 293T cells were transfected with HA-ATDC. 24hr post transfect ion, cells were treated with 1.3uM TSA overnight. Anti-HA immunoprecipitates obtained under low st ringency conditions were analyzed by West ern blotting with p53 antibody DO-1. (C) U2OS cells were transfected as indicated. 24hr post transfection, cells were treated with ethanol carrier or 1.3uM TSA overnight. Cells were harvested and luciferase activities were determined in triplicate. Error bars show standard deviations. (D) U2OS cells were transfected or infected as i ndicated. 48hr post transfection or infection, cells were harvested, and cell lysates were analyzed as indicated. might not play an important role in regul ating ATDC function. However, K116 in ATDC was quite well conserved, suggesting it might be an impor tant functional acetylation site. To test this idea, human ATDC K102 and K116 were mutated to arginine, individually. As shown in figure 16, mutating lysine 116 to argine significantly decrease ATDC-p53 complex formation and p53 cytoplasm sequestration (Figure 16B). Ho wever, there is no effect by mutating human lysine 102 to arginine. Further, K116R mutant co uld abolish the ATDC inhibitory effect on p53 transcriptional activity (Figure 16C). Interestingly, K116R mutant also abolish the ability of ATDC to complement the radiosensitivity of AT5BIVA cells, but not by K102R mutant (Figure 16D). These data suggested that ATDC biological function was dynam ically controlled through K 116 acetylation. However, the function of other acetylation site s of ATDC is still not understood yet. ATDC was Overexpressed in Pancr eatic and Cervical Carcinoma It was reported that mRNA of ATDC was significantly amplif ied in pancreatic carcinoma and cervical carcinoma (Iacobuz io-Donahue et al., 2002; Zhang et al.

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71 A B ATDC p53 Merged wt K102R K116R

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C 0200000400000600000800000100000012000001400000160000018000002000000vectorWTK116R 72 Figure 16. ATDC K116 acetylation play a major role in regulating ATDC-p53 interaction (A) Alignment of ATDC homologous including human, chimpanzee, mouse, rat and dog. (B) U2OS cells were transfected as indicated. 36hr post transfection, cells were fixed, permeabilized, and stained with anti-HA and anti-p53 DO-1 antibody, P<0.05 P<0.05 HA-ATDC K116R + HA-ATDC WT + HA-ATDC WT/K116R IB anti-HA p53 IB anti-p53 actin IB anti-actin 1 2 3 D 051015 2025% Survival 303540VectorWTK102RK116R

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73 followed by Alexa-555 and Alexa-488-conjugated secondary an tibodies. Samples were analyzed using Zeiss Confocal Microscopy. (C) U2OS cells were transfected as indicated. 36hr post transfection, cells were harvested and luciferase activities were determined in triplicate. Error bars show standard deviations. Statistical analysis was done by student t test. Fractions of lysates were analyzed by western blot to check protein expression. (D) AT5BIVA cells transfected with plasmids (4ug each) encoding t he indicated proteins were irradiated (2Gy) or left unirradiated. The surviving colonies were counted two weeks later. 2005). As previous shown in figure 13C, AT DC protein is also highly expressed in pancreatic carcinoma tissue. Similarl y, the ATDC protein is also highly expressed in the pancreatic carcinoma cell lines that we tested, including Hs766T and BxPC3, and cervical carcinom a cell lines includ ing HeLa and SiHa (Figure 17A) compared to other tumor ce ll lines. By immunostaining study, we found endogenous ATDC colocalized with endogenous p53 in the cytoplasm in SiHa cell line (wild-type p53+/+) (Fi gure 17B). Neverthel ess, RNAi-mediated reduction of endogenous ATDC leads to nuclear localization of p53 in SiHa cells (Figure 17C). Fruthermore, we tested whether shRNA-mediated inhibition of ATDC function could radio-sensitize cervic al carcinomal cells. As indicated in figure 17D, native SiHa cervical carci noma cells responded poorly to treatment with -irradiation. However, treatment wit h ATDC shRNA significantly makes the cells more susceptible to irradiation treatment. SiHa cells were also sensitized by overexpression of HDAC9, probably through antagonizing the endogenous ATDC function. Furthermore, shRNA-mediated reduction of ATDC and forcedexpression of HDAC9 have an additive effe ct on SiHa cell radiosensitivity when treated with high dosage of -irradiation (7.5Gy) (Figure 17D). Thus, the combination of knocking-down ATDC ex pression and introduction of HDAC9

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Pancreatic carcinoma Cervical Carcioma Cervical Carcioma 74 ATDC Actin 1 2 3 4 5 6 7 8 9 10 11 Colorectal Cancer Kidney Epithelial Osteosarcoma Lung Carcioma Pancreatic Carcinoma A ATDC p53 Merged B C 02040 60 801000Gy2.5Gy7.5Gy Control SiRNA ATDC SiRNA Ad-GFP Ad-HDAC9 Control SiRNA+Ad-GFP ATDC SiRNA+Ad-HDAC9 % Survival

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75 Figure 17. Endogenous ATDC in cervical carcinoma cells regulates p53 nuclear localization and function (A) ATDC is highly expressed in pancreatic and cervical carcinoma cell lines. Whol e-cell extracts of different cell lines were immunoblotted with anti-ATDC antibody (upper) and anti-acti n antibody (lower). (1.HCT116; 2. U2OS; 3. Hs766T; 4. CAPAN-2; 5. Bx-PC3; 6. AT5BIVA; 7. HeLa; 8. 293T; 9. Si Ha; 10. A549; 11. COLO357). (B) SiHa cells were fixed, permeabilized, and stained with anti-ATDC and anti-p53 DO -1 antibodies, followed by Alexa-555 and Alexa-488conjugated secondary antibodies, and analyzed us ing Zeiss Confocal Microscopy. (C) SiHa cells were transfected with control or ATDC siRNA. 48 hr post transfection, cells we re fixed, permeabilized, and st ained with antiATDC and anti-p53 DO-1 antibodies, followed by Alexa-555 and Alexa-488-c onjugated secondary antibodies, and analyzed using Zeiss Confocal Microscopy. (D) SiHa cells transfected or infected as indicated. 48hr post transfection or infection, cells were irradiated (2.5 or 7.5Gy) or left unirradiated. The su rviving colonies were counted 10 days later. might be an alternative treat ment for the radio-resi stant tumors caused by amplified ATDC expression. Growth Stress Induce ATDC Acetylation Since ATDC plays an important role in regulating p53-m ediated apoptotic pathway and might have a significant effe ct on the clinical radio-therapy for tumors, it is tempting to identify the physiological conditions under which ATDC are highly acetylated in cells. We treated cervical carcinoma cells with various extracellular stimuli and performed West ern blot experiments with antibodies to acetylated lysine. As shown in figur e 18A and 18B, acetylation of endogenous ATDC in HeLa cells is dynamically induc ible in response to growth factors stimulation including EGF (Figure 18A) and PMA (Figure 18B). In contrast, ATDC acetylation was not affected by -irradiation (data not shown). Thus, ATDC acetylation appears to be specifically induced in response to growth stress stimuli. Similar results were obtained in SiHa cells tr eated with PMA (Figure 18C).

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76 EGF 100ng/ml (min) 0 10 15 30 60 120 unstarve A Ac-ATDC IP ATDC IB AcK IgG H IP ATDC ATDC IB ATDC IgG H 1 2 3 4 5 6 7 IP ATDC IB AcK IP ATDC IB ATDC Ac-ATDC ATDC PMA 50ng/ml (min) 0 2 5 10 15 30 60 90 unstarve B IgG H IgG H 1 2 3 4 5 6 7 8 9 C unstarve PMA 50ng/ml (min) 0 5 10 15 30 60 IP ATDC Ac-ATDC IB AcK IgG H ATDC IP ATDC IB ATDC IgG H 1 2 3 4 5 6 7

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D ATDC PMA 50ng/ml p53 Merged 0min 10min 30min 60min Figure 18. Growth stress regulates ATDC acetylation (A) HeLa cells were starved for 18hr, and treated with EGF (100ng/ml) or left untreated as indicated. Cell lysates were then immunoprecipitated under high stringency conditions with anti-ATDC antibodies. Endogenous acetylated ATDC was analyzed by Western blotting with a mixture of polyclonal anti-acetyl-lysine antibodies. (B) HeLa cells were starved for 77

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78 18hr, and treated with PMA (50ng/ml) or left untreated as indi cated. Acetylation of endogenous ATDC was analyzed as in Panel A. (C) SiHa cells were starved for 18hr, and treated with PMA (50ng/ml) or left untreated as indicated. Acetylation of endogenous ATDC was analyzed as in Panel A. (D) SiHa cells were starved for 18hr, and treated with PMA (50ng/ml) or left untreated as indicated. Cells were fixed, permeabilized, and stained with anti-ATDC and anti-p53 DO-1 antibodies, followed by Alexa-555 and Alexa-488-conjugated secondary anti bodies, and analyzed using Zeiss Confocal Microscopy. Interestingly, we found that ATDC interacts with p53 in SiHa cell cytoplasm in a growth stress-inducible manner (Figur e 18D), which could be correlated with growth stress-induced ATDC acetylati on (Figure 18C). These data suggested that ATDC acetylation, which could be regulated by growth factors in tumor microenvironment, might be helpful for tumor cells to resist ionizing radiation therapy and survive. Dissussion The present data reveal that ATDC, th e product of the ataxia-telangiectasia group D complementing gene, is acetylat ed dynamically within the cells in a growth stress stimulation manner. And HD AC9 and p300 acetyltransferase as its interaction partners play important roles to balance ATDC reversible acetylation. Importantly, ATDC is a cytoplasmic protein that is critically involved in the regulation of p53 subcellular localiz ation and subsequent function in cells. HDACs play critical roles in diverse biological functions (Ruijter et al., 2003; Marks et al., 2003). The HDAC9 mutant mice displays an enhanced response to stress signals and enhanced hypertrophic phenotype (Zhang et al., 2002), suggesting this HDAC acts in the adult hear t to suppress the fetal gene program and hypertrophic growth. And this functi on was carried out by forming a complex

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79 with an important transcription factor MEF2 (the myocyte enhancer-binding factor 2) (Zhang et al., 2002; Haberland et al., 2007) In this study, using immunoaffinity purification method, we i dentify another impor tant HDAC9 inte raction partner ATDC. Through preventing the ATDC-p5 3 complex formation, HDAC9 could sensitize the tumor cells with a radi o-resistant phenotype caused by abnormal expression of ATDC. Thes e data would have an important implication for future clinical radio-therapy for cancer. The molecular basis of the defect in AT is still largely unknown, although the diversity of phenotypes has led to t he suggestion that a signaling mechanism may be defective. ATDC, which complements the ionizing radiation sensitivity of AT group D cell lines, might involve in a signal transduction pathway that is induced by ionizing radiation. It has been previously repor ted that it could physically interact with vimentin, a type III intermediate filament; with hPKCI-1, a small protein inhibitor of PKC (Brzoska et al., 1995). Some experiments data suggested aberrant p53 function may be a majo r factor in the hy persensitivity of AT cells to ionizing radiation (Meyn et al ., 1994). In the curr ent study, ATDC was found it could physically interact and colocalize with p53 in the cell cytoplasm. By controlling p53 subcellular localization and its subsequent biological function, ATDC could facilitate the cell survival under -irradiation stress. Taken together, these data suggest a model in which ATDC -p53 complex involves in an ionizing radiation-induced signal transduction pathway.

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80 As a transcription factor, nuclear localizat ion of p53 is essentia l for its role in tumor suppression (Vousden et al., 2002; Jimenez et al., 1999). However, p53 is diffusely distributed in the cytoplasm of normal unstressed cells; many types of tumors cells have abnormal cytoplasmic localization of p53 and an impaired p53dependent stress response despite the fact that they express wild-type p53 proteins (Jimenez et al., 1999; Moll et al., 1996). In previous stud ies, it has been shown that the majority of cytoplasmic p53 is tightly associated with endogenous Parc (p53-associated, Parkin-like cytoplas mic protein) (Nikolaev et al., 2003). In the absence of stress, inactivation of endogenous Parc leads p53 activation through inducing p53 nuclear localization. In current study, ATDC was also found has a similar role as Parc in regulat ing p53 function. Furthermore, ATDC-p53 complex formation is regulated by ATDC K116 acetylation, which is controlled by HDAC9. And our results also indicate t hat the levels of ATDC proteins are relatively high in cervical and pancreatic carcinoma cell lines and pancreatic tumor tissues. It is conceivable, therefore, that the high levels of ATDC in these cells may prevent nuclear translocation of p53, even in the presence of genotoxic stress although other mechanisms may also contribute to this phenomenon. In consistence with this notion, we have al so shown that shRNA-mediated reduction of ATDC protein levels or introduction of HDAC9 can restore a strong p53dependent stress response in cervical carc inoma SiHa cells. As such, the ATDCmediated pathway of p53 regulat ion may prove to be a potential target for cancer therapy. In particular, agents that down-r egulate ATDC protein levels or abrogate

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81 the ATDC-p53 interaction, such as intr oduction of HDAC9, may sensitize tumor cells to p53-dependent apoptosis. As an acetylated protein, p53 function was directly regulated by a class I HDAC, HDAC1 (Luo et al., 2000), and a class III HDAC, SIRT1 (Luo et al., 2001; Vaziri et al., 2001). In current study, we report a class II HDAC, HDAC9 could indirectly regulate p53-mediated apoptot ic pathway by regulating its interaction with ATDC. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function. References 1. Aizawa, H., Hu, S.C., Bobb, K., Bala krishnan, K., Ince, G., Gurevich, I., Cowan, M., and Ghosh, A. (2004). Dendrite development regulated by CREST, a calcium-regulated transcriptiona l activator. Science 303, 197-202. 2. Brzoska, P.M., Chen, H., Zhu, Y., Levin N.A., Disatnik, M.H., Mochly-Rosen, D., Murnane, J.P., Christman, M.F. (1995). The product of the ataxiatelangiectasia group D complementing gene, ATDC, interact s with a protein kinase C substrate and inhibitor. Proc Natl Acad Sci U S A. 92(17):7824-8. 3. Chan, H.M., Krstic-Demonac os, M., Smith, L., Demo nacos, C., La Thangue, N.B. (2001). Acetylation control of th e retinoblastoma tumour-suppressor protein. Nat Cell Biol. 3(7):667-74. 4. Chong, M. J., Murray, M.R ., Gosink, E.C ., Russell, H.R ., Srinivasan, A ., Kapsetaki, Korsmeyer, S.J ., McKinnon, P.J (2000). Atm and Bax cooperate

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ABOUT THE AUTHOR Zhigang Yuan was born in Shanxi, China in a family of 3 children. After 5 years study at the medical college of Jilin university (the former Norman Bethune university of medical sciences), Changch un, China, Mr. Yuan received his bachelor degree of medicine in the summe r of 1999. He went on pursuing his master degree in the department of immunology at the medical center of Fudan university (the former Shanghai medical university), Shanghai, China from August, 1999 to June, 2002. In August, 2002, Mr. Yuan was enrolled in the Ph.D. program in the department of molecular medicine at the University of South Floridas college of medici ne, Tampa, Florida. While enrolling in the Ph.D. program, the American Heart Associat ion granted Mr. Yuan a two-year predoctoral fellowship for his innovative research proposal.


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Functional characterization of roles of histone deacetylases in the regulation of DNA damage response
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ABSTRACT: Histone deacetylases (HDACs) are a family of enzymes whose functions have been overwhelmingly associated with gene expression and chromatin dynamics by modifying the histone tails. In recent years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activities have been found to be regulated by posttranslational acetylation on the -amino group of lysines. Here, we report that two DNA repair factors including NBS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Complementing) are acetylated proteins. SIRT1 could maintain NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.
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