||Characterization of ZBRK1 gene regulation and function in DNA damage response
||Institute of Basic Medical Sciences
視網膜母细胞瘤蛋白（retinoblastoma protein, RB）除了具有重要的腫瘤抑制功能，也被認為在DNA損傷所導致的細胞週期停滯中扮演重要的角色，且可調控許多與DNA修復相關的因子。然而在DNA損傷反應中，RB如何藉由轉錄機制調控與生長停滯相關的基因仍不是非常清楚。我們探討在DNA損傷反應中，RB是否可以藉由調控ZBRK1這個可以調控DNA損傷反應中重要基因GADD45A的鋅手指的轉錄因子，進而導致細胞週期的停滯。實驗結果發現ZBRK1基因啟動區包含一個會專一與E2F1結合，但不會與E2F4和E2F6結合的E2F結合序列，同時更發現RB, CtIP, CtBP與E2F1會形成複合體，在UV及MMS刺激下共同抑制ZBRK1基因轉錄。除此之外，我們證明了即使ZBRK1的結合蛋白BRCA1及KAP1在DNA損傷時會累積在DNA損傷位置，ZBRK1本身並不會出現在DNA損傷位置。表示ZBRK1主要透過調控下游基因的轉錄調控而參與在DNA損傷的反應中。進一步研究結果中也發現，缺乏ZBRK1表現的會導致 GADD45A及ANG1表現量增加，且使細胞受到UV及MMS刺激時不會產生大量的DNA損傷累積。綜上所述，我們的研究結果證明RB/E2F1複合體在抑制ZBRK1轉錄機制上扮演重要角色，並且當此抑制功能失去時，會使得DNA損傷累積導致血管新生以及癌症的形成。
Despite the critical importance of retinoblastoma protein (RB) function in tumor suppression, RB also has an essential role in DNA damage-induced growth arrest and regulates the expression of several factors essential for DNA repair machinery. However, how RB coordinates DNA damage response through transcriptional regulation of genes involved in growth arrest remains largely unexplored. We examined whether RB can mediate the response to DNA damage through modulation of ZBRK1, a zinc finger-containing transcriptional repressor that can modulate the expression of GADD45A, a DNA damage response gene, to induce cell cycle arrest in response to DNA damage. We found that ZBRK1 promoter contains an authentic E2F-recognition sequence that specifically binds E2F1, but not E2F4 or E2F6. Moreover, through this E2F motif, E2F1 can interact with RB, CtIP, and CtBP to form a complex for repressing ZBRK1 gene transcription upon UV and MMS treatment. Alternatively, we demonstrated that ZBRK1 does not localize to the DNA damage foci, even its interaction proteins BRCA1 and KAP1 have been shown to bind these foci. This suggests that ZBRK1-mediated DNA damage response may majorly through a transcription-dependent manner. Furthermore, loss of ZBRK1 expression in cells can result in GADD45A and ANG1 gene activation and shows a resistant effect in UV- and MMS-induced DNA damage. Taken together, these results suggest that the RB/E2F1 complex plays a critical role in ZBRK1 transcriptional repression and loss of this repression may contribute to the sensitivity of DNA damage insults intimately linked to angiogenesis and carcinogenesis.
Abbreviation List ix
Chapter 1 Introduction
1.1 The DNA damage response 1
1.1.2 The DNA damage inducers 1
1.1.3 The DNA repair mechanism 2
1.2 Zinc finger protein (ZFP) 3
1.2.1 Krüpple-associated box-containing zinc-finger proteins (KRAB–ZFPs) 4
1.2.2 Zinc finger and BRCA1-interacting protein with a KRAB domain-1 (ZBRK1) 4
1.2.3 The role of ZBRK1 in DNA damage response 5
1.3 The RB/E2F pathway 6
1.3.1 Retinoblastoma protein (RB) 7
1.3.2 E2F-family proteins 8
1.3.3 The role of RB in DNA damage response 9
1.4 The DNA damage and angiogenesis 10
1.4.2 The role of ZBRK1 in angiogenesis 10
1.5 Specific aims 11
Chapter 2 Materials and methods
2.1 Materials 12
2.2 Methods 12
Cell Culture and Treatment of DNA Damage Agents 12
Plasmid Construction 13
Transfection and Reporter Gene Assay 13
Reverse Transcription Polymerase Chain Reaction (RT-PCR) 14
Western Blot and DNA Affinity Precipitation Assay (DAPA) 14
Chromatin immunoprecipitation (ChIP) and re-ChIP Assay 15
Comet Assay 16
Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay 16
Lentiviral shRNA 17
Microscopy and UVA-laser Irradiation 17
Chapter 3 Results
RB protein level is inversely associated with ZBRK1 transcript level. 18
The E2F recognition site identified on the ZBRK1 promoter is biologically active. 18
E2F1, but not E2F4 or E2F6, acts as a negative regulator of the ZBRK1 promoter. 19
RB is crucial for E2F1-mediated repression of ZBRK1 reporter activity. 20
RB acts as a negative regulator of the ZBRK1 promoter through the E2F motif. 20
Interaction of CtIP with CtBP is important for RB/E2F1-mediated repression of ZBRK1 reporter activity. 21
E2F1/Rb/CtIP/CtBP complex binds to the E2F motif of ZBRK1 promoter. 21
Loss of RB can induce ZBRK1 expression and results in an increase in the DNA damage response. 22
Half-life of endogenous ZBRK1 is shorter than that of exogenous GFP-ZBRK1. 23
E2F1/RB/CtIP/CtBP-mediated ZBRK1 repression is involved in both UV- and MMS-induced DNA damage response. 23
ZBRK1 does not localize to the DNA damage foci. 24
Depletion of ZBRK1 results in an increase in GADD45A expression. 24
Depletion of ZBRK1 protects cells from UV- and MMS-induced DNA damage. 25
Loss of ZBRK1 upon DNA damage response is associated with angiogenesis related genes. 25
ZBRK1 and KAP1 lose the repressive effect on ANG1 transcription after UV treatment. 26
Chapter 4 Discussion 28
Curriculum Vitae 68
Ahmed, K.M., Tsai, C.Y. and Lee, W.H. (2010) Derepression of HMGA2 via removal of ZBRK1/BRCA1/CtIP complex enhances mammary tumorigenesis. J Biol Chem, 285, 4464-4471.
Almasan, A., Yin, Y., Kelly, R.E., Lee, E.Y., Bradley, A., Li, W., Bertino, J.R. and Wahl, G.M. (1995) Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis. Proc Natl Acad Sci U S A, 92, 5436-5440.
Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M.B., Bartek, J. and Lukas, J. (2006) Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol, 173, 195-206.
Bellefroid, E.J., Poncelet, D.A., Lecocq, P.J., Revelant, O. and Martial, J.A. (1991) The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc Natl Acad Sci U S A, 88, 3608-3612.
Bosco, E.E., Mayhew, C.N., Hennigan, R.F., Sage, J., Jacks, T. and Knudsen, E.S. (2004) RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult. Nucleic Acids Res, 32, 25-34.
Brehm, A., Miska, E.A., McCance, D.J., Reid, J.L., Bannister, A.J. and Kouzarides, T. (1998) Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature, 391, 597-601.
Campisi, J. and d'Adda di Fagagna, F. (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol, 8, 729-740.
Chen, C.F., Li, S., Chen, Y., Chen, P.L., Sharp, Z.D. and Lee, W.H. (1996) The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J Biol Chem, 271, 32863-32868.
Chung, J.H. and Eun, H.C. (2007) Angiogenesis in skin aging and photoaging. J Dermatol, 34, 593-600.
Croxton, R., Ma, Y., Song, L., Haura, E.B. and Cress, W.D. (2002) Direct repression of the Mcl-1 promoter by E2F1. Oncogene, 21, 1359-1369.
Dang, D.T., Pevsner, J. and Yang, V.W. (2000) The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol, 32, 1103-1121.
Dasika, G.K., Lin, S.C., Zhao, S., Sung, P., Tomkinson, A. and Lee, E.Y. (1999) DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene, 18, 7883-7899.
Debnath, J., Muthuswamy, S.K. and Brugge, J.S. (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods, 30, 256-268.
DeGregori, J., Leone, G., Miron, A., Jakoi, L. and Nevins, J.R. (1997) Distinct roles for E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci U S A, 94, 7245-7250.
Deng, C.X. and Wang, R.H. (2003) Roles of BRCA1 in DNA damage repair: a link between development and cancer. Hum Mol Genet, 12 Spec No 1, R113-123.
Doisneau-Sixou, S.F., Sergio, C.M., Carroll, J.S., Hui, R., Musgrove, E.A. and Sutherland, R.L. (2003) Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells. Endocr Relat Cancer, 10, 179-186.
Elledge, S.J. (1996) Cell cycle checkpoints: preventing an identity crisis. Science, 274, 1664-1672.
Feuerhahn, S. and Egly, J.M. (2008) Tools to study DNA repair: what's in the box? Trends Genet, 24, 467-474.
Field, S.J., Tsai, F.Y., Kuo, F., Zubiaga, A.M., Kaelin, W.G., Jr., Livingston, D.M., Orkin, S.H. and Greenberg, M.E. (1996) E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell, 85, 549-561.
Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M. and Dryja, T.P. (1986) A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 323, 643-646.
Furuta, S., Wang, J.M., Wei, S., Jeng, Y.M., Jiang, X., Gu, B., Chen, P.L., Lee, E.Y. and Lee, W.H. (2006) Removal of BRCA1/CtIP/ZBRK1 repressor complex on ANG1 promoter leads to accelerated mammary tumor growth contributed by prominent vasculature. Cancer Cell, 10, 13-24.
Garcia, V., Garcia, J.M., Pena, C., Silva, J., Dominguez, G., Rodriguez, R., Maximiano, C., Espinosa, R., Espana, P. and Bonilla, F. (2005) The GADD45, ZBRK1 and BRCA1 pathway: quantitative analysis of mRNA expression in colon carcinomas. J Pathol, 206, 92-99.
Hanway, D., Chin, J.K., Xia, G., Oshiro, G., Winzeler, E.A. and Romesberg, F.E. (2002) Previously uncharacterized genes in the UV- and MMS-induced DNA damage response in yeast. Proc Natl Acad Sci U S A, 99, 10605-10610.
Harbour, J.W. and Dean, D.C. (2000) The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev, 14, 2393-2409.
Harrington, E.A., Bruce, J.L., Harlow, E. and Dyson, N. (1998) pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc Natl Acad Sci U S A, 95, 11945-11950.
Hofferer, M., Wirbelauer, C., Humar, B. and Krek, W. (1999) Increased levels of E2F-1-dependent DNA binding activity after UV- or gamma-irradiation. Nucleic Acids Res, 27, 491-495.
Hurford, R.K., Jr., Cobrinik, D., Lee, M.H. and Dyson, N. (1997) pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. Genes Dev, 11, 1447-1463.
Inoue, Y., Kitagawa, M. and Taya, Y. (2007) Phosphorylation of pRB at Ser612 by Chk1/2 leads to a complex between pRB and E2F-1 after DNA damage. EMBO J, 26, 2083-2093.
Jager, A.C., Rasmussen, M., Bisgaard, H.C., Singh, K.K., Nielsen, F.C. and Rasmussen, L.J. (2001) HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes. Oncogene, 20, 3590-3595.
Johnson, D.G. (2000) The paradox of E2F1: oncogene and tumor suppressor gene. Mol Carcinog, 27, 151-157.
Johnson, D.G., Schwarz, J.K., Cress, W.D. and Nevins, J.R. (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature, 365, 349-352.
Jun, D.Y., Park, H.S., Lee, J.Y. and Kim, Y.H. (2008) Regulation of the human mitotic centromere-associated kinesin (MCAK) promoter by the transcription factors Sp1 and E2F1. Biochim Biophys Acta, 1779, 356-361.
Khan, M.Z., Brandimarti, R., Shimizu, S., Nicolai, J., Crowe, E. and Meucci, O. (2008) The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein. Cell Death Differ.
Koipally, J. and Georgopoulos, K. (2002) Ikaros-CtIP interactions do not require C-terminal binding protein and participate in a deacetylase-independent mode of repression. J Biol Chem, 277, 23143-23149.
Kosmoski, J.V., Ackerman, E.J. and Smerdon, M.J. (2001) DNA repair of a single UV photoproduct in a designed nucleosome. Proc Natl Acad Sci U S A, 98, 10113-10118.
Lan, L., Nakajima, S., Oohata, Y., Takao, M., Okano, S., Masutani, M., Wilson, S.H. and Yasui, A. (2004) In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proc Natl Acad Sci U S A, 101, 13738-13743.
Li-Fang Lin, C.-H.C., Chien-Feng Li, Ching-Chun Liao, Chun-Pei Cheng, Tian-Lu Cheng, Meng-Ru Shen, Joseph T. Tseng, Wen-Chang Chang, Wen-Hwa Lee, and Ju-Ming Wang (2010) ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer. Cancer Res, 70, 192-201.
Liao, G., Huang, J., Fixman, E.D. and Hayward, S.D. (2005) The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor. J Virol, 79, 245-256.
Lin, L.F., Chuang, C.H., Li, C.F., Liao, C.C., Cheng, C.P., Cheng, T.L., Shen, M.R., Tseng, J.T., Chang, W.C., Lee, W.H. and Wang, J.M. (2010) ZBRK1 acts as a metastatic suppressor by directly regulating MMP9 in cervical cancer. Cancer Res, 70, 192-201.
Liu, F. and Lee, W.H. (2006) CtIP activates its own and cyclin D1 promoters via the E2F/RB pathway during G1/S progression. Mol Cell Biol, 26, 3124-3134.
Looman, C., Abrink, M., Mark, C. and Hellman, L. (2002) KRAB zinc finger proteins: an analysis of the molecular mechanisms governing their increase in numbers and complexity during evolution. Mol Biol Evol, 19, 2118-2130.
Lundin, C., North, M., Erixon, K., Walters, K., Jenssen, D., Goldman, A.S. and Helleday, T. (2005) Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Res, 33, 3799-3811.
Meloni, A.R., Smith, E.J. and Nevins, J.R. (1999) A mechanism for Rb/p130-mediated transcription repression involving recruitment of the CtBP corepressor. Proc Natl Acad Sci U S A, 96, 9574-9579.
Miller, J., McLachlan, A.D. and Klug, A. (1985) Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. Embo J, 4, 1609-1614.
Morgenbesser, S.D., Williams, B.O., Jacks, T. and DePinho, R.A. (1994) p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature, 371, 72-74.
Muller, H., Bracken, A.P., Vernell, R., Moroni, M.C., Christians, F., Grassilli, E., Prosperini, E., Vigo, E., Oliner, J.D. and Helin, K. (2001) E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev, 15, 267-285.
Ohtani-Fujita, N., Fujita, T., Takahashi, R., Robbins, P.D., Dryja, T.P. and Sakai, T. (1994) A silencer element in the retinoblastoma tumor-suppressor gene. Oncogene, 9, 1703-1711.
Ohtani, K., DeGregori, J. and Nevins, J.R. (1995) Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci U S A, 92, 12146-12150.
Pan, H., Yin, C., Dyson, N.J., Harlow, E., Yamasaki, L. and Van Dyke, T. (1998) Key roles for E2F1 in signaling p53-dependent apoptosis and in cell division within developing tumors. Mol Cell, 2, 283-292.
Pascucci, B., Russo, M.T., Crescenzi, M., Bignami, M. and Dogliotti, E. (2005) The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells. Nucleic Acids Res, 33, 280-288.
Peters, K.G. (1998) Vascular endothelial growth factor and the angiopoietins: working together to build a better blood vessel. Circ Res, 83, 342-343.
Pickering, M.T. and Kowalik, T.F. (2006) Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene, 25, 746-755.
Putzer, B.M. (2007) E2F1 death pathways as targets for cancer therapy. J Cell Mol Med, 11, 239-251.
Ramji, D.P. and Foka, P. (2002) CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J, 365, 561-575.
Sage, J., Mulligan, G.J., Attardi, L.D., Miller, A., Chen, S., Williams, B., Theodorou, E. and Jacks, T. (2000) Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev, 14, 3037-3050.
Schaeper, U., Subramanian, T., Lim, L., Boyd, J.M. and Chinnadurai, G. (1998) Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A (CtBP) and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif. J Biol Chem, 273, 8549-8552.
Sewalt, R.G., Gunster, M.J., van der Vlag, J., Satijn, D.P. and Otte, A.P. (1999) C-Terminal binding protein is a transcriptional repressor that interacts with a specific class of vertebrate Polycomb proteins. Mol Cell Biol, 19, 777-787.
Sherr, C.J. (2000) The Pezcoller lecture: cancer cell cycles revisited. Cancer Res, 60, 3689-3695.
Shi, Y., Sawada, J., Sui, G., Affar el, B., Whetstine, J.R., Lan, F., Ogawa, H., Luke, M.P., Nakatani, Y. and Shi, Y. (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature, 422, 735-738.
Sinha, R.P. and Hader, D.P. (2002) UV-induced DNA damage and repair: a review. Photochem Photobiol Sci, 1, 225-236.
Stevaux, O. and Dyson, N.J. (2002) A revised picture of the E2F transcriptional network and RB function. Curr Opin Cell Biol, 14, 684-691.
Subramanian, T. and Chinnadurai, G. (2003) Association of class I histone deacetylases with transcriptional corepressor CtBP. FEBS Lett, 540, 255-258.
Tan, W., Kim, S. and Boyer, T.G. (2004) Tetrameric oligomerization mediates transcriptional repression by the BRCA1-dependent Kruppel-associated box-zinc finger protein ZBRK1. J Biol Chem, 279, 55153-55160.
Urrutia, R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol, 4, 231.
Wang, J.M., Tseng, J.T. and Chang, W.C. (2005) Induction of human NF-IL6beta by epidermal growth factor is mediated through the p38 signaling pathway and cAMP response element-binding protein activation in A431 cells. Mol Biol Cell, 16, 3365-3376.
Wang, W., Smith, R., 3rd, Burghardt, R. and Safe, S.H. (1997) 17 beta-Estradiol-mediated growth inhibition of MDA-MB-468 cells stably transfected with the estrogen receptor: cell cycle effects. Mol Cell Endocrinol, 133, 49-62.
Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S.J. and Qin, J. (2000) BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev, 14, 927-939.
Weinberg, R.A. (1995) The retinoblastoma protein and cell cycle control. Cell, 81, 323-330.
Weintraub, S.J., Prater, C.A. and Dean, D.C. (1992) Retinoblastoma protein switches the E2F site from positive to negative element. Nature, 358, 259-261.
Wu, L., Goodwin, E.C., Naeger, L.K., Vigo, E., Galaktionov, K., Helin, K. and DiMaio, D. (2000) E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression. Mol Cell Biol, 20, 7059-7067.
Wu, L., Timmers, C., Maiti, B., Saavedra, H.I., Sang, L., Chong, G.T., Nuckolls, F., Giangrande, P., Wright, F.A., Field, S.J., Greenberg, M.E., Orkin, S., Nevins, J.R., Robinson, M.L. and Leone, G. (2001) The E2F1-3 transcription factors are essential for cellular proliferation. Nature, 414, 457-462.
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E. and Dyson, N.J. (1996) Tumor induction and tissue atrophy in mice lacking E2F-1. Cell, 85, 537-548.
Yun, J. and Lee, W.H. (2003) Degradation of transcription repressor ZBRK1 through the ubiquitin-proteasome pathway relieves repression of Gadd45a upon DNA damage. Mol Cell Biol, 23, 7305-7314.
Zheng, L., Pan, H., Li, S., Flesken-Nikitin, A., Chen, P.L., Boyer, T.G. and Lee, W.H. (2000) Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1. Mol Cell, 6, 757-768.
Zhou, B.B. and Elledge, S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature, 408, 433-439.
Ziv, Y., Bielopolski, D., Galanty, Y., Lukas, C., Taya, Y., Schultz, D.C., Lukas, J., Bekker-Jensen, S., Bartek, J. and Shiloh, Y. (2006) Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol, 8, 870-876.