Identification of PARP1 as a transcriptional regulator of HBV replication

Identification of PARP1 as a Transcriptional Regulator of HBV Replication Ko Hui Ling (Gao Huiling) B.Sc. (Hon) National University of Singapore A Thesis submitted for the degree of Doctor of Philosophy National University of Singapore 2010 ACKNOWLEDGEMENTS I thank my supervisor, Prof Ren, for his constant encouragement and immense support, for giving me the freedom to develop my ideas and providing invaluable guidance when in doubt. I also thank him for his patience in listening to my problems and providing sound advice to the problems I have faced. I thank my friend Chi Hsien, who taught me to fight for my passion and pursue a career in science. I thank my husband Wen Chun, for all the love and support he has given me and his kind understanding of how important this work means to me. I thank him for cheering me up through the most difficult times of my life. I thank my parents for teaching me how to face the hardships in life so that I could handle the hurdles experienced with an open-mind. I further thank them for adjusting their lifestyle to the nature of my work, so that we may have a happy dinner together even when my experiments stretch late into the night. I thank my sisters for staying up with me, so that I may not feel alone when analyzing my results. They are a constant source of delight to pull me through the dark times. To my friends, Ziwei, Zhi Ying, Emily, Pey Yng and Meixin, thank you bringing joy and constant laughter. I would also like to thank Hui Jun and Ming Keat for technical assistance, and Wang Bei and Stanley for technical guidance. And I thank A*STAR (Agency for Science, Technology and Research) for supporting my work. I Contents TABLE OF CONTENTS Summary…………………………………………………………………………………… V List of tables…………………………………………………………………………….….VII List of figures………………………………………………………………………….… .VIII List of illustrations………………………………………………………………………… .X List of abbreviations…………………… .…………………………………………………XI Chapter Introduction 1.1 The hepatitis B virus………………………………………………………2 1.2 PARP1 and its functions…………………………………………………12 1.3 Outline and aims………………………………………………………….29 Chapter Variability of HBV replication in cell lines 2.1 Introduction……………………………………………………………… .33 2.2 The replicative HBV construct………………………………………… .35 2.3 Differential replication efficiencies of HBV in cell lines………… … .41 2.4 Conclusion…………………………………………………………………46 II Contents Chapter PARP1 is a novel transcriptional activator at the HBV core promoter 3.1 Introduction…………………………………………………………….… 48 3.2 Screen for critical transcriptional regulators……………………………50 3.3 Novel transcriptional activator has uncharacterized motif……………61 3.4 PARP1 binds HBV core promoter in sequence-specific manner…… 73 3.5 PARP1 is required for efficient HBV replication……………………….86 3.6 PARP1 enzymatic inhibition enhances HBV replication…………… 90 3.7 Conclusion…………………………………………………………….….104 Chapter Aberrant PARP1 binding motif expression impairs DNA damage repair 4.1 Introduction…………………………………………………….…………107 4.2 Defining the PARP1 binding motif………………………….………….109 4.3 PARP1 inhibition and impaired DNA repair by motif binding……….117 4.4 HBV genotype C possesses extra copy of PARP1 binding motif …128 4.5 PARP1 motif enhances cytotoxicity of DNA damage inducers….….133 4.6 The PARP1 binding motif as a novel class of PARP1 inhibitor…….139 4.7 Conclusion……………………………………………………………… 146 III Contents Chapter Discussion 5.1 PARP1 enzymatic activity and the PARP1-HBVCP interaction…….148 5.2 The PARP1 binding motif as a novel therapeutic……………………157 5.3 Novel therapeutic strategies against HBV infections……………… 165 5.4 PARP1 V762A polymorphism, HBV replication and HCC………….168 Chapter Materials and methods…………………………………………………174 References…………………………………………………………………………………I Appendix A: List of publications and manuscripts…………………………………… A Appendix B: Submitted PNAS manuscript….…………………………… .………… B IV Summary SUMMARY There are 350 million chronic carriers of the hepatitis B virus (HBV) worldwide who face increased risks of developing liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). The severity of disease is associated with high replication efficiency of HBV, which is in turn dependent on the establishment of functional hostpathogen interactions, as factors such as HBV pathogen genotype and host factor variability are predictors of infection outcomes. HBV infection is currently treated by boosting the immune system to aid viral clearance or inhibition of HBV polymerase function with nucleoside or nucleotide analogues. These have their limitations—the former is only efficacious in certain individuals while the latter have resulted in the generation of drug-resistant strains. Importantly, both are incapable of eliminating the virus. Therefore, there is a need to design novel strategies for the treatment of chronic HBV infection. The synthesis of infectious HBV particles depends on the activity of host binding factors regulating transcription at the HBV core promoter (HBVCP). Modulating the function of such transcription factors thus seems an obvious solution for combating HBV infection. Since infection outcomes differ greatly among individuals, it was hypothesized that subtle differences in these factors contribute significantly to the efficacy of HBV replication hence infection outcome. To understand how transcription at the HBVCP may be differentially regulated, a screen was performed at the HBVCP for binding factors in four cell lines whose ability to support HBV replication differ. The results show that only a handful of described host factors critically affect transcription at the HBVCP, including SP1, hnRNP K and HNF1, all of which serve vital functions in cells hence renders them unfavourable as therapeutic targets. The screen also discovered a novel binding site for a transcriptional activator that did not correspond to any previously known factors V Summary and this was subsequently shown to bind poly (ADP-ribose) polymerase (PARP1), an enzyme involved in DNA repair and transcriptional regulation. Not only was PARP1 important for transcriptional activation at the HBVCP, its enzymatic activity was found to inversely correlate with the efficiency of HBV replication. This led to the discovery that a polymorphic variant with low enzymatic activity often expressed in HBV endemic areas accounts for high HBV replication efficiency. Since the ablation of PARP1 activity is not known to be lethal, PARP1 is a favourable therapeutic candidate for the treatment of chronic HBV infection. Even though PARP1 is known to be a transcription factor, its recognition motif has not been defined. This was resolved by studying how individual nucleotides contribute to transcription at the PARP1 binding site of the HBVCP. Surprisingly, in contrast to enzymatic activation by binding DNA strand-breaks during DNA repair, binding the PARP1 motif results in enzymatic inhibition. Exogenous expression of the PARP1 binding motif was sufficient to reduce cellular PARP1 enzymatic activity, leading to impaired DNA repair hence cytotoxicity with DNA damage induction. This was reproducible with replicative HBV, providing a mechanism for the association of high viral load and DNA damage accumulation leading to HCC. The novel phenomenon achieved by the PARP1 motif puts it in a new class of PARP1 inhibitors with therapeutic potential. This is the first report that PARP1 is an important transcriptional regulator in HBV replication. In addition, by studying the HBVCP, the PARP1 consensus binding motif was uncovered. The PARP1 binding motif was further demonstrated to inhibit PARP1 enzymatic activity. Not only is this useful for cancer therapy, it also providing insights into the role of HBV in the development of HCC. VI List of Tables Title Page HBV products and their functions Treatment of chronic HBV infection 11 PARP1 associated diseases and outcomes of enzymatic inhibition 25 Properties of liver-derived cell lines used 42 Guidelines for indentifying therapeutic targets that bind the HBVCP 53 Nucleotide positions critical for PARP1 motif binding 161 Comparing the PARP1 binding motif and general PARP inhibitors 163 List of RT-PCR primers 175 List of primers for generating HBVCP deletion mutants 177 10 Sequences of cloning primers 179 11 Primer sequences for detecting HBV cccDNA and HBV transcripts 183 12 List of 20bp DNA duplexes used in histone H1 modification assays 184 VII List of Figures Title Page HBV envelope proteins expression and secretion 37 Generation of replicative HBV with the HBV-RFP construct 41 HBV transcripts may only be expressed in certain cell lines 43 Differential capabilities in expressing different HBV products 45 Effect of URR deletions on the HBV core promoter 55 Prediction of transcription factors binding to URR17 62 Expression of HNF4α in different cell lines 66 HNF4α is not the URR17-binder 69 Oct-1 does not bind URR17 sequence 72 10 PARP1 is novel transcriptional activator at HBV core promoter 74 11 PARP1 binds the HBV core promoter in a sequence dependent manner 76 12 PARP1 regulates transcription at HBVCP in motif dependent manner 77 13 PARP1 regulates pgRNA and pcRNA synthesis 81 14 PARP1 expression and knockdown 87 15 Effect of PARP1 knockdown on HBV replication 89 16 PARP1 inhibition enhances PARP1 dependent transcription 92 17 PARP1 inhibition increase HBV replication 94 18 PARP1 is expressed in all cell lines used 95 19 HepG2 has low endogenous PARP1 enzymatic activity 96 20 HepG2 and Huh-6 express the V762A mutant PARP1 98 21 The V762A polymorphism and PARP1 function 100 22 The PARP1 V762A polymorphism supports HBV replication 102 23 Allelic frequency of the V762A SNP extracted from dbSNP 103 24 The PARP1 recognition motif 110 25 Confirmation of the PARP1 motif in different cell lines 113 VIII List of Figures Title Page 26 Describing validated PARP1 binding sites with novel PARP1 motif 114 27 The PARP1 binding site is highly conserved across HBV genotypes 116 28 Inhibition of PARP1 enzymatic activity by binding PARP1 motif 118 29 HBV PARP1 binding motif impairs DNA damage repair 121 30 PARP1 binding motif alone sensitizes cells to induced DNA damage 124 31 HBx protein does not impair DNA damage repair 125 32 Sensitivity to DNA damage with PARP1 motif is PARP1 specific 127 33 Full-length genome of HBV genotype C results in greater DNA damage 130 34 HBV genotype C has additional copy of PARP1 binding motif 132 35 Increased apoptosis with HBV replication by induced DNA damage 135 36 HBV mediated sensitization to etoposide induced cell death in Huh-7 136 37 PARP1 motif enhances cytotoxicity by DNA damage inducers 137 38 Enhanced apoptosis by PARP1 motif is PARP1 dependent 138 39 Comparing the effect of PARP1 motif with clinical PARP inhibitors 142 40 Suppression of HBV replication with PARP1 motif 144 IX Submitted manuscript 16. D'Amours D, Desnoyers S, D'Silva I, Poirier GG. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J 1999;342 ( Pt 2):249-268. 17. Henning SM, Swendseid ME, Coulson WF. Male rats fed methyl- and folate-deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly(ADP-ribose) polymerase activity. J Nutr 1997;127:30-36. 18. Shall S, de Murcia G. Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model? Mutat Res 2000;460:1-15. 19. Tong WM, Cortes U, Hande MP, Ohgaki H, Cavalli LR, Lansdorp PM, Haddad BR, et al. Synergistic role of Ku80 and poly(ADP-ribose) polymerase in suppressing chromosomal aberrations and liver cancer formation. Cancer Res 2002;62:6990-6996. 20. H, Shibata A, Kamada N, Masumura K, Nohmi T, Kobayashi S, Teraoka H, Nakagama et al. Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent. Oncogene 2005;24:1328-1337. 21. Shibata A, Maeda D, Ogino H, Tsutsumi M, Nohmi T, Nakagama H, Sugimura T, et al. Role of Parp-1 in suppressing spontaneous deletion mutation in the liver and brain of mice at adolescence and advanced age. Mutat Res 2009;664:20-27. 22. Dandri M, Burda MR, Burkle A, Zuckerman DM, Will H, Rogler CE, Greten H, et al. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation. Hepatology 2002;35:217-223. 23. Kramvis A, Kew MC. The core promoter of hepatitis B virus. J Viral Hepat 1999;6:415-427. 24. Moolla N, Kew M, Arbuthnot P. Regulatory elements of hepatitis B virus transcription. J Viral Hepat 2002;9:323-331. 25. Hong P, Ng LF, Ren EC, Chen WN. A cell-based system for hepatitis B virus replication: significance of clinically enhanced viral replication in relation to deletions in viral core promoter. Front Biosci 2005;10:2001-2004. 26. Ng LF, Chan M, Chan SH, Cheng PC, Leung EH, Chen WN, Ren EC. Host heterogeneous ribonucleoprotein K (hnRNP K) as a potential target to suppress hepatitis B virus replication. PLoS Med 2005;2:e163. S Submitted manuscript 27. Kraus WL. Transcriptional control by PARP-1: chromatin modulation, enhancer- binding, coregulation, and insulation. Curr Opin Cell Biol 2008;20:294-302. 28. Krishnakumar R, Kraus WL. The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets. Mol Cell 2010;39:8-24. 29. Simbulan-Rosenthal CM, Ly DH, Rosenthal DS, Konopka G, Luo R, Wang ZQ, Schultz PG, et al. Misregulation of gene expression in primary fibroblasts lacking poly(ADPribose) polymerase. Proc Natl Acad Sci U S A 2000;97:11274-11279. 30. Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985;317:489-495. 31. Nassal M, Schaller H. Hepatitis B virus replication. Trends Microbiol 1993;1:221-228. 32. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of cccDNA function in hepatitis B virus infection. J Hepatol 2009;51:581-592. 33. Huambachano O, Herrera F, Rancourt A, Satoh MS. Double-stranded DNA Binding Domain of Poly(ADP-ribose) Polymerase-1 and Molecular Insight into the Regulation of Its Activity. J Biol Chem 2011;286:7149-7160. 34. Zhang Z, Hildebrandt EF, Simbulan-Rosenthal CM, Anderson MG. Sequence-specific binding of poly(ADP-ribose) polymerase-1 to the human T cell leukemia virus type-I tax responsive element. Virology 2002;296:107-116. 35. Ohsaki E, Ueda K, Sakakibara S, Do E, Yada K, Yamanishi K. Poly(ADP-ribose) polymerase binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency. J Virol 2004;78:9936-9946. 36. Smith S. The world according to PARP. Trends Biochem Sci 2001;26:174-179. 37. Kameoka M, Nukuzuma S, Itaya A, Tanaka Y, Ota K, Inada Y, Ikuta K, et al. Poly(ADP-ribose)polymerase-1 is required for integration of the human immunodeficiency virus type genome near centromeric alphoid DNA in human and murine cells. Biochem Biophys Res Commun 2005;334:412-417. 38. Lockett KL, Hall MC, Xu J, Zheng SL, Berwick M, Chuang SC, Clark PE, et al. The ADPRT V762A genetic variant contributes to prostate cancer susceptibility and deficient enzyme function. Cancer Res 2004;64:6344-6348. T Submitted manuscript 39. Figueroa JD, Malats N, Real FX, Silverman D, Kogevinas M, Chanock S, Welch R, et al. Genetic variation in the base excision repair pathway and bladder cancer risk. Hum Genet 2007;121:233-242. 40. Huang K, Tidyman WE, Le KU, Kirsten E, Kun E, Ordahl CP. Analysis of nucleotide sequence-dependent DNA binding of poly(ADP-ribose) polymerase in a purified system. Biochemistry 2004;43:217-223. 41. Pottier N, Cheok MH, Yang W, Assem M, Tracey L, Obenauer JC, Panetta JC, et al. Expression of SMARCB1 modulates steroid sensitivity in human lymphoblastoid cells: identification of a promoter SNP that alters PARP1 binding and SMARCB1 expression. Hum Mol Genet 2007;16:2261-2271. 42. Ambrose HE, Papadopoulou V, Beswick RW, Wagner SD. Poly-(ADP-ribose) polymerase-1 (Parp-1) binds in a sequence-specific manner at the Bcl-6 locus and contributes to the regulation of Bcl-6 transcription. Oncogene 2007;26:6244-6252. 43. Jagtap P, Szabo C. Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors. Nat Rev Drug Discov 2005;4:421-440. 44. Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG. PARP inhibition: PARP1 and beyond. Nat Rev Cancer;10:293-301. 45. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 2006;7:517-528. ACKNOWLEDGEMENTS We thank Prof W.N. Chen (Nanyang Technological University) for the kind gift of the HBV replicon. We also thank M.K. Sng for technical assistance; B. Wang, Z. Xiao and members of the E.C.R. lab and the Protein and Proteomics Center (National University of Singapore) for technical help and advice. U Submitted manuscript FIGURE LEGENDS Fig. 1. PARP1 is a novel transcriptional activator at the HBVCP. (A) Selective enrichment of an approximately 120 kDa protein using μg of the biotinylated HBVCP probe (5’TTGAGGCCTACTTCAAAGACTGTGTGT-3’) in affinity pull-down assays from 70 μg HepG2 nuclear lysates. The MALDI-TOF/TOF result identifies PARP1 as the specific binder. Sequenced peptides are underlined and bold. (B) ng of biotinylated HBVCP probe was incubated with μg HepG2 nuclear lysates treated with non-specific siRNA. The binding complex formed in the presence of μg poly-dIdC could be removed with 100-fold excess of unlabeled probe, PARP1 specific antibody or with the use of nuclear lysates with PARP1 specific knock-down (K/D). The relative amounts of PARP1 in the nuclear lysates used are shown. (C) Deduction of the putative PARP1 binding site by luciferase reporter assays. The HBVCP fragment (nt 1600-1860) was cloned upstream of a luciferase reporter construct; five constructs were generated in which each contained a 15 bp deletion that overlaps to span nt 1689-1727. Results show normalized mean luminescence ± SE relative to the wild-type promoter of three independent experiments performed in triplicates. The exact nucleotides deleted in each construct are represented by gray lines. Fig. 2. The PARP1 recognition motif. The effect of all possible single base substitutions on the predicted PARP1 core motif “TTCAAA” and the bases flanking either of its sides were determined by examining altered luciferase expression. Results show mean normalized luminescence ± SE of triplicates relative to that of the wild-type promoter. Relative positions of each nucleotide with respect to the defined PARP1 binding motif are indicated. Fig. 3. HBV replication is dependent on PARP1. (A) HBV replication in HepG2 is indicated by the production of HBV surface antigens in cytoplasmic lysates and the accumulation of cccDNA with time. (B) Effect of PARP1 specific knock-down on HBV replication as indicated by cccDNA fold-change relative to hours post-transfection. Values represent mean foldchange ± SE of independent experiments performed in triplicates. ***P < 0.001 with one-tailed V Submitted manuscript student’s t-test. (C) Effect of PARP1 specific knock-down on enhancer II function as determined by immunofluorescence (IF) staining for HBs expression 60 hours after siRNA treatment. HBV-RFP transfected cells fluoresce red. Fig. 4. PARP1 motif reduces PARP1 enzymatic activity in vitro. The effect of PARP1 binding to 20 bp DNA duplexes containing the PARP1 motif “ACATCAAA” (highlighted) on PARP1 enzymatic activity was investigated by histone H1 modification assays. Mutations to the duplex sequence are shown in bold. The effect of PARP1 specific knock-down (K/D) on PARP1 activity is also shown for comparison to demonstrate that the near absence of PARP1 expression reduces in vitro histone H1 modification by 40%. Means of an experiment ± SE performed in triplicates are shown. **P < 0.01 using one-tailed student’s t-test. Fig. 5. Exogenous PARP1 motif impairs cellular DNA repair. (A) Inhibition of PARP1 dependent histone H1 modification by short DNA duplexes containing the “ACTTCAAA” HBVCP PARP1 binding motif. The mean ± SE reduction in histone H1 modification of triplicates with the viral PARP1 motif relative to buffer control was analyzed using one-tailed student’s t-test with equal variance. ***P < 0.001. (B) Construct containing the HBVCP PARP1 binding motif “ACTTCAAA” in tandem repeats. (C) The extent of DNA damage visualized by alkaline comet assays induced with 50 nM etoposide and 20 ng/ml bleomycin in HepG2 cells transfected PARP1 motif construct of independent experiments is as plotted and analyzed using one-tailed student’s t-test. Cells transfected with empty vector were used as controls. Bars indicate the median extent of DNA damage. ***P < 0.001 with one-tailed t-test (D) HBVCP PARP1 motif sensitizes cells to apoptosis by DNA damaging agents. Dying cells stain positive by annexin V staining resulting from irreparable DNA damage induced by 50 nM etoposide or 20 ng/ml bleomycin. 0.01% DMSO and empty vectors were used as controls. Fig. 6. PARP1 motif dependent enhanced cytotoxicity to induced DNA damage is PARP1 specific. Exogenous PARP1 was readily detectable in 10 μg of HepG2 nuclear lysates within 48 hours of transfection with μg of its expression plasmid. μg of the plasmid containing the W Submitted manuscript PARP1 binding motif and 500 ng of the PARP1 over-expression plasmid were co-transfected into HepG2 cells, and the mean percentage ± SE increase in caspase activity associated with the PARP1 motif relative to the empty vector was compared to that of RFP over-expression. DNA damage was induced by 100 nM etoposide or 10 ng/ml bleomycin of independent experiments. ***P < 0.001 obtained using one-tailed student’s t-test. O/E, over-expression. SUPPORTING INFORMATION Supporting Information Fig. 1. Generation of luciferase assay constructs with 15 bp deletions. (A) Schematic of relative positions of primers used to generate 5’ and 3’ sequences flanking of each deletion. (B) Primer sequences used to generate each flanking sequence. Supporting Information Fig. 2. A second, independent experiment of an affinity pull-down assay to identify the bound complex was analyzed by MALDI-TOF/TOF. The results provide additional confirmatory data (Fig. 1A) demonstrating the specificity of PARP1 for the HBVCP probe. Supporting Information Fig. 3. PARP1 binds the HBVCP in a sequence dependent manner. Large excess of 1000-fold poly-dIdC cannot diminish the PARP1 specific complex, whereas 100-fold of unlabeled HBVCP probe was sufficient to so. Supporting Information Fig. 4. The PARP1 binding motif is conserved in the HBVCP. Gen A = Genotype A; n = number of aligned sequences. Supporting Information Fig. 5. The HBV-RFP construct. 1.1-fold of the viral genome was inserted upstream of the CMV promoter, thus viral replication depended only on host factors that bind the HBVCP (CP). A short stretch of the replicon, including the HBVCP and the polyadenylation signal needs to be duplicated to ensure the production of all viral transcripts. X Submitted manuscript Constitutive expression of red fluorescent protein (RFP) driven by the CMV promoter was used to show transfection into cells. Supporting Information Fig. 6. PARP1 specific knockdown can be achieved within 24 hours of transfecting PARP1 specific siRNA. The effects of the PARP1 specific siRNA can last at least 48 hours. Supporting Information Fig. 7. Inhibition of PARP1 function by exogenous PARP1 binding motif. Co-transfection of μg of the HBV-RFP with 500 ng of the plasmid containing the HBVCP PARP1 binding motif results in a reduction of enhancer II function as shown by a reduction in HBs expression in HepG2 cells 60 hours post-transfection. Cells co-transfected with HBV-RFP and empty vector (pcDNA) were used as controls. Y Submitted manuscript Supporting Information Table 1. Primer sequences for cloning and quantitative real-time PCR. Cloning primer Primer sequence PGLF 5’-TCCCCAGTGCAAGTGCAGG-3’ PGLR 5’-TTTGGCGTCTTCCATGGTGGC-3’ HBVF 5’-TGCCAATTGTTTACGCGGTCTCCCC-3’ HBVR 5’-TGCACGCGTGCTCCAAATTCTTTATAAGG-3’ PARP1motif-F 5’-AATTGTACTTCAAAGTACTTCAAAGTACTTCAAAGA-3’ PARP1motif-R 5’-CGCGTCTTTGAAGTACTTTGAAGTACTTTGAAGTAC-3’ PARP1-F 5’-TTAGCTAGCATGGCGGAGTCTTCGG-3’ PARP1-R 5’-AAGCTCGAGCTACCTCTCCCAATTACC-3’ Real-time PCR primer Primer sequence cccF 5’-GCACCTCTCTTTACGCGGTCTCC-3’ cccR 5’-TGAAGCGAAGTGCACACGGACCG-3’ pcDNA-F1 5’-TGGATAGCGGTTTGACTCACGGGG-3’ pcDNA-R1 5’-ATTTGCGTCAATGGGGCGGAGTTG-3’ Z Submitted manuscript Supporting Information Table 2. Sequences of 20 bp DNA duplexes used in histone H1 modification assays. DNA duplex Sense strand Antisense strand 5’-ACGTCTACATCAAAGTTGCA-3’ 5’-TGCAACTTTGATGTAGACGT-3’ 5’-CCGTCTACATCAAAGTTGCA-3’ 5’-TGCAACTTTGATGTAGACGG-3’ 5’-CTATCTACATCAAAGTTGCA-3’ 5’-TGCAACTTTGATGTAGATAG-3’ 5’-ACGTCTACATCCAAGTTGCA-3’ 5’-TGCAACTTGGATGTAGACGT-3’ 5’-ACGTCTACATGCAAGTTGCA-3’ 5’-TGCAACTTGCATGTAGACGT-3’ 5’-ACGTCTACAGGCAAGTTGCA-3’ 5’-TGCAACTTGCCTGTAGACGT-3’ 5’-ACGTCTACTTCAAAGTTGCA-3’ 5’-TGCAACTTTGAAGTAGACGT-3’ Wild-type PARP1motif “CC” mutation flanking motif “CTA” mutation flanking motif “C” mutation within motif “GC” mutation within motif “GGC” mutation within motif HBVCP PARP1 motif AA Submitted manuscript Supporting Information Table 3. Relative PARP1 mRNA expression in paired tumor and nontumor samples. Paired sample PARP1 expression Tumor/ Non-tumor HCC93 HCC171 HCC188 HCC100 HCC159 HCC174 HCC182 HCC98 HCC112 HCC111 HCC63 HCC128 HCC13 HCC155 HCC130 HCC69 HCC107 HCC85 HCC86 HCC154 HCC77 HCC124 HCC103 HCC102 HCC83 HCC163 HCC101 HCC99 HCC105 HCC67 HCC168 HCC96 HCC110 HCC81 HCC118 HCC90 HCC64 0.98 0.99 1.00 1.01 1.04 1.05 1.05 1.06 1.06 1.06 1.07 1.08 1.08 1.09 1.09 1.10 1.11 1.12 1.12 1.12 1.12 1.13 1.13 1.14 1.14 1.14 1.14 1.15 1.16 1.17 1.18 1.18 1.19 1.19 1.19 1.20 1.21 Mean 1.11 SD 0.06 BB Su bmitted ma anuscript Figure Figure CC C Submitted ma anuscript Figure Figure DD D Submitted ma anuscript Figure Figure EE Submitted ma anuscript Supporting g Informatiion Figure g Informatiion Figure Supporting g Informatiion Figure Supporting FF Submitted ma anuscript Supporting g Informatiion Figure g Informatiion Figure Supporting GG G Submitted ma anuscript Supporting g Informatiion Figure Supporting g Informatiion Figure HH H [...]... yet characterized The extensive PAR network on activated PARP1 is also an important cue for the assembly of chromatin remodeling complexes, DNA repair machinery and DNA damage check-point proteins such as ATM (Ataxia Telangiectasia Mutated)152 Importantly, PAR on auto-modified PARP1 need not be synthesized by activated PARP1, as PAR can be added onto PARP1 by other members of the PARP family such as the... transcription at HBVCP 149 17 Potential roles of PARP1 enzymatic activity in HBV replication 154 18 Rhythmic cycling of PARP1 enzymatic activity and HBV replication 156 19 PARP1 binding motif as a novel class of PARP1 inhibitors 162 20 Comparing therapeutic strategies against HBV 166 21 Factors contributing to variable outcomes of HBV infection 169 22 PARP1 V76 2A polymorphism, HBV replication and HCC 171... formation of DNA strand breaks, hence alkylating agents such as NMU (N-nitroso-N-methylurea) and DNA cross-linkers such as cisplatins can also result in the activation of PARP1 Thus, PARP1 is a key regulator of DNA repair as several types of DNA lesions to be rectified depend on its activity DNA strand-breaks need not be formed as a result of DNA damage, but may be purposefully induced to enhance genetic... transcript and protein expression180 However, due to the apparent lack of similarity in aligned PARP1 binding sites, its motif remains elusive PARP1 in Disease States and Effect of PARP1 Inhibition With PARP1 playing multiple important roles such as DNA repair and transcriptional regulation, it is not surprising that altered PARP1 activity and expression has been associated with several disease states (Table... otypes of HBV9 Gen notype C pre-domina ant in the East Asian continen and is clinically nt c associated with poorer prognos and hig sis gher incidence of HC 10 This may be CC attributed t higher ga to ain -of- funct tion mutatio rates6 as well as in on s ncreased viral load and DNA9 Genotype F though r rare is foun mainly in Central a nd and South America A and associated with fu ulminant he epatitis and HCC11,... DNA repair complex PARP1 R PARP1 dissociation w Low cellular NA + pool AD N 2 Apo optosis 3 N Necrosis Illustration 10 Role of PARP1 in DNA re n epair PARP binds D P1 DNA strand breaks d induced by various DN damagin agents This activate its enzym NA ng T es matic activit hence ty ication, the resultant P PAR acts as a cue to attract DNA repair com s a A mplexes auto-modifi Continued auto-modification... phosphatase/ kinase), the gap-filling polymerase DNA polymerase β and DNA ligase III Automodified PARP1 also recruits nucleosome repositioning proteins such as ALC1 (Amplified in Liver Cancer 1) to make damaged DNA accessible for repair128, 164 At stalled replication forks, the activated and auto-modified PARP1 recruits another set of proteins including Mre11127 18 Intro oduction N DNA d damaged R N Irradiatio... binds DNA Illustration 9 PARP1 modification, activity modulation and its effects Casp— Caspase; TF—Transcription factor; NA—Nicotinamide analogues 15 Introduction Perhaps the best studied PARP1 activator is nicked DNA PARP1 enzymatic activity may also be stimulated by interacting partners such as phosphorylated ERK2144, 145 Post-translational modifications and interactions with inhibitory proteins add... conserved amongst PARPs whose function has not been characterized106 It has been proposed that this is another DNA binding domain N Zn I Zn II NLS Casp NLS Dimerization DNA-binding Zn III PADR1 BRCT WGR Auto-modification Reg Catalytic domain C Catalytic domain Illustration 7 Functional domains of PARP1 Zn—Zinc-binding domains; NLS— Nuclear localization signal; Casp—Caspase cleavage site; BRCT—BRCA1 C Terminus... DNA A affinity of P 19 Introduction Even though PARP1 is required for the assembly of DNA repair or replication restart machinery, it also poses steric hindrance by binding at the site of DNA damage Therefore, when PAR gets too extensive, the accumulation of negative charges on PARP1 forces its repulsion from DNA to allow DNA repair or DNA replication re-start to proceed The release of PARP1 from DNA . enzymatic activity in HBV replication 154 18 Rhythmic cycling of PARP1 enzymatic activity and HBV replication 156 19 PARP1 binding motif as a novel class of PARP1 inhibitors 162 20 Comparing. genome of HBV genotype C results in greater DNA damage 130 34 HBV genotype C has additional copy of PARP1 binding motif 132 35 Increased apoptosis with HBV replication by induced DNA damage 135. Identification of PARP1 as a Transcriptional Regulator of HBV Replication  Ko Hui Ling (Gao Huiling) B.Sc. (Hon) National University of Singapore A Thesis