INVESTIGATING THE ROLES OF BRD2, BRD4, DEK, AND MeCP2 IN THE DNA SEGREGATION MECHANISM OF THE EPSTEIN-BARR VIRUS
by
Ammy Lin
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Molecular Genetics
University of Toronto
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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada Investigating the Roles of Brd2, Brd4, DEK and MeCP2 in the DNA Segregation
Mechanism of the Epstein-Barr Virus
by Ammy Lin
Masters of Science, Graduate Department of Molecular Genetics,
University of Toronto, 2008
ABSTRACT
Epstein-Barr Nuclear Antigen-1 (EBNA1) mediates the stable segregation of Epstein-Barr Virus
(EBV) genomes by tethering them to host mitotic chromosomes. Previously, our laboratory showed that EBV-based plasmid maintenance can be reconstituted in Saccharomyces cerevisiae in the presence of EBNA1 and human EBNAl-binding protein-2 (hEBP2). I investigated the ability of cellular proteins DEK, Brd2, Brd4 and MeCP2, previously implicated in the maintenance of other extrachromosomal viral genomes, to perform similar roles in EBNA1- mediated segregation as hEBP2. I found that Brd4 conferred plasmid maintenance in an
EBNA1-dependent fashion. Plasmid partitioning by Brd4 required the N-terminus of EBNA1 to which Brd4 was also shown to bind in human cells. Silencing Brd4 did not affect EBNA1 association with mitotic chromosomes, suggesting that the segregation role of Brd4 in human cells may be minor. However, a correlation between Brd4 binding and transcriptional activity of
EBNA1 suggested that Brd4 may mediate transcriptional activation by EBNA1. ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisor, Dr. Lori Frappier, whose enthusiasm for science inspired me to pursue research when I was a fourth-year student. I am thankful for her guidance over the years, her patience, great ideas and support. I would also like to thank my committee members, Dr. Brigitte Lavoie and Dr. Brent Deny, for their great ideas, critical comments, and support. I would especially like to acknowledge my brilliant yeast guru, Bri, for telling me funny stories that contained meaningful advice, for giving me endless help and encouragement, and whose optimism and love for science always kept me motivated and enthusiastic. I am also grateful to all the members of the Frappier lab, both past and present: Kathy Shire, Nirojini Sivachandran, Jayme Salsman, Amos Sakwe and Priya Kapoor. Thank you all for your help and support. The lab would not have been nearly as enjoyable without you guys. I would especially like to thank Vipra Nayyar, Shan Wang, Tin Nguyen, Feroz Sarkari and Teresa Sanchez. You guys not only taught me about science, but also about life. You were my personal cheerleading squad, support system, psychiatrist and voice of reason. I would have never survived without you! Thank you! To my friends who always believed in me, supported my decisions, and visited me in the lab despite their busy schedules: Arany Shan, Kit Man Wong, Savil Hemati, May Khanna, Christine Misquitta, Matthew Chuk, Lori Ing and Anna Wong. Those coffee breaks and meals that we shared helped keep my sanity. Thank you for your friendship. Last but certainly not least, I would like to thank my family whose unconditional love, moral support and encouragement helped me to achieve this Masters degree. Mom, Dad, Jennie, Annie and Polly: you guys have always stood at the front lines with me, listened to my complaints, celebrated my triumphs and coped with all my highs and lows. With you by my side, I could achieve anything. I thank you from the bottom of my heart. I love you all!
in TABLE OF CONTENTS
ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF TABLES viii LIST OF ABBREVIATIONS ix
I. INTRODUCTION 1
1.1. The Epstein-Barr Virus 1 1.1.1. Infection and Associated Diseases 1 1.1.2. Latent Infection and Viral Gene Expression 2 1.1.3. Viral Origin of Latent Replication (oriP) 3 I.1.3.a.DS Element 4 I.1.3.b.FR Element 6 1.1.4. EBNA1 Functions and Domains 7 1.1.4.a. EBNA1 Functional Domains and Structure 7 I.1.4.b. EBNA1 Functions 10 1.1.4.b.i. Latent EBV Episome Replication 11 1.1 Ab.ii. Transcriptional Activation 13 1.1 Ab.iii. Segregation of the EBV Episome and EBV-Based Plasmids 14 I.lAb.iv. Cell Immortalization 16 1.2. Mechanism of EBV Segregation 17 1.2.1. The Mitotic Tethering or "Hitchhiking" Model of EBV 17 1.2.2. The Role of hEBP2 in EBNA1-Mediated Segregation of EBV-Based Plasmids 18 1.2.2.a. Properties and Cellular Function of EBP2 19 1.2.2.b. Interaction Between EBNA1 and human EBNA1-Binding Protein 2(hEBP2) 21 I.2.2.C. hEBP2 Reconstitutes EBNA1 -Mediated Plasmid Maintenance in Budding Yeast 22 I.2.2.d. hEBP2 Plays an Important Role in EBNA1-Mediated Segregation in Human Cells 24 1.2.2.e. EBP2 is Regulated by Aurora Family Kinases 25 I.2.2.f. The Role of hEBP2 in the Mitotic Tethering Model of EBV 26 1.2.3. Even Distribution of EBV Episomes on Sister Chromatids Leads to Equal Partitioning 26 1.3. Mechanism of Segregation of Other Viruses 29 1.3.1. Bovine Papillomavirus Type-1 (BPV1) 29 1.3.2. Kaposi's Sarcoma-Associated Herpesvirus (KSHV) 34 1.3.3. Herpesvirus Saimiri (HVS) 40 1.4. Thesis Rationale 41
iv II. MATERIALS AND METHODS 43
II. 1. Plasmid Loss Assay Constructs 43 II.2. Plasmid Loss Assay 44 II. 3. Co-Immunoprecipitation Constructs 47 11.4. Co-Immunoprecipitation in 293T Cells 48 11.5. Silencing Constructs for hBrd4 (esiBRD4), hBrd2 (siBRD2) an hMeCP2 (siMeCP2) 49 11.6. Silencing hBrd4, hBrd2 and hMeCP2 Inside HeLa.EBNAl Cells 50 11.7. Biochemical Fractionation 51 11.8. Antibodies 52 11.9. SDS-PAGE and Western Blot Analysis 52 II. 10. Checking Protein Expression in Budding Yeast 53
III. RESULTS 54
III. 1. hEBP2 is Able to Reconstitute EBV-Based Plasmid Maintenance in YPH499 54 111.2. DEK Does Not Promote FR Plasmid Maintenance in Budding Yeast 59 111.3. Brd2 and MeCP2 are General Mediators of Plasmid Segregation 59 111.4. mBrd4 Confers FR Plasmid Stability in the Presence of EBNA1 63 111.5. The N-terminus of EBNA1 is Required for mBrd4-Mediated Segregation of the FR Plasmid 66 111.6. The Transactivation Domain of EBNA1 Mediates an Interaction between EBNA1 and hBrd4 68 111.7. EBNA1 Remains Associated with Mitotic Chromosomes Upon Brd4 Down- Regulation 72 III. 8. EBNA1 Remains Associated with Interphase Chromatin Upon Brd4 Down- Regulation 75 III. 9. EBNA1 Remains Associated with Cellular Chromatin Upon Brd2 and MeCP2 Down-Regulation 76
IV. DISCUSSION 79
IV. 1. DEK is Unable to Mediate FR Plasmid Maintenance in Yeast 79 IV.2. Brd2 and MeCP2 are General Mediators of Plasmid Maintenance 80 IV.3. The Role of Brd4 in EBNA1-Mediated Segregation in Budding Yeast 84 IV.4. The Role of Brd4 in Segregation in Human Cells 89 IV.5. Brd4 May Have Multiple Roles in the EBV Life Cycle 93 IV.6. Assessing the Ability of EBNA1 to Bind DNA Throughout the Cell Cycle 94 IV.7. Multiple Pathways and Mechanisms to Support EBV Episome Partitioning 95
V. REFERENCES 98
v LIST OF FIGURES
Figure 1 The EBV latent origin of replication, oriP 5
Figure 2 Schematic diagram of the structural and functional domains of EBNA1 8
Figure 3 Schematic diagram of the structural and functional domains of hEBP2 20
Figure 4 hEBP2 and EBNA1-mediated mitotic tethering model of EBV 27
Figure 5 The proposed mitotic tethering models of BVP1, KSHV and HVS 30
Figure 6 The experimental protocol for the yeast plasmid loss assay 55
Figure 7 Segregation test plasmids, controls, and test conditions used in the yeast
plasmid loss assay 56
Figure 8 hEBP2 reconstitutes EBV-Based plasmid maintenance in YPH499 58
Figure 9 DEK Does Not Promote FR Plasmid Maintenance in Budding Yeast 60
Figure 10 Brd2 is a general mediator of plasmid maintenance 61
Figure 11 MeCP2 is a general mediator of plasmid maintenance 62
Figure 12 Protein sequence alignment of human and mouse Brd4 64
Figure 13 mBrd4 reconstitutes EBV-Based plasmid maintenance in YPH499 65
Figure 14 The N-terminus of EBNA1 is required for mBrd4-mediated segregation of the FR plasmid in yeast 67 Figure 15 Schematic diagram of EBNA1 mutants tested in the co-immunoprecipitation (co-IP) experiments 69
Figure 16 EBNA1 binds Brd4 and EBNA1 residues 61-83 are required for the interaction 71
Figure 17 Silencing Brd4 does not affect EBNA1 fractionation with chromatin in G2 or mitosis 74
Figure 18 Silencing Brd4 does not affect EBNA1 fractionation with chromatin during logarithmic growth 77
VI Figure 19 Silencing Brd2 or MeCP2 does not affect EBNA1 fractionation with chromatin throughout the cell-cycle
Figure 20 Different domains of EBNA1 are required for hEBP2 versus mBrd4- mediated segregation of the FR plasmid in yeast
Figure 21 Multiple proposed mechanisms of EBV segregation
vii LIST OF TABLES
Table 1 Yeast strains used in this study 45
Table 2 A summary of the effects of EBNA1 and EBNA1 mutants on mBrd4- mediated segregation of the FR plasmid in yeast and their interactions with Brd4 in human cells 87
Vlll LIST OF ABBREVIATIONS
2D Two dimensional AIDS Acquired immunodefiency syndrome ARS Autonomously replicating sequence AT Adenine-thymine Bdflp Bromodomain factor 1 protein Bdf2p Bromodomain factor 2 protein BL Burkitt's lymphoma bp Base pairs BPV1 Bovine papillomavirus type-1 Brd2 Bromodomain protein 2 Brd4 Bromodomain protein 4 BRLF1 BamHI C leftward frame 1 BZLF1 BamHI Z leftward frame 1 CAS 1/2 Chromosome association site 1 or 2 CAT Chloramphenicol acetyltransferase CBP Ca2+/cAMP response element binding protein (CREB) Binding Protein CEN Centromeric ChIP Chromatin immunoprecipitiation ChlRl chromosome loss-related protein 1 CMV Cytomegalovirus Co-IP Co-immunoprecipitation CpG Cytosine-phosphate-guanine CREB cAMP Response Element CTD C-terminal domain DBDR DNA-binding and dimerization region DNA Deoxyribonucleic acid dNTPs Deoxynucleotide triphosphate DS Dyad symmetry EBER Epstein-Barr expressed ribonucleic acid
IX EBNA1 Epstein-Barr nuclear antigen 1 EBP2 EBNA1-binding protein 2 EBV Epstein-Barr virus ET Extra-terminal FISH Fluorescence in-situ hybridization FR Family of repeats GFP Green fluorescent protein Gly-Ala Glycine-alanine Gly-Arg Glycine-arginine GRG Glycine-arginine-glycine HAUSP/USP7 Herpes-associated ubiquitin specific protease/ubiquitin specific protease 7 hBrd4 Human bromodomain protein 4 hEBP2 Human EBNA1-binding protein 2 HHV Human herpesvirus HMG-1 High mobility group-1 HVS Herpesvirus saimiri IE Immediate early IM Infectious mononucleosis Ipll Increase-in-ploidy protein 1 Kb Kilobase kD Kilodalton KSHV Kaposi's sarcoma-associated herpesvirus LANA1 Latency-associated nuclear antigen-1 LCLs Lymphoblastoid cell lines LCV Lymphocryptovirus Leu Leucine LiAc Lithium acetate LMP Latent membrane protein MBD Methyl binding domain mBrd4 Mouse bromodomain protein 4 MCM Minichromosome maintenance
X MeCP2 Methyl CpG-binding protein 2 MHC Major histocompatibility complex MME Minichromosome maintenance element MO Minimal origin of replication Napl Nucleosome assembly protein 1 NLS Nuclear localization signal NPC Nasopharyngeal carcinoma OD Optical density ORC Origin recognition complex ORF Open reading frame oriLyt Origin of lytic replication oriP Origin of plasmid replication PBS Phosphate buffered saline PCR Polymerase chain reaction PKK Proline-lysine-lysine pRB Retinoblastoma protein RGG Arginine-glycine-glycine RPA Replication protein A rRNA Ribosomal ribonucleic acid SC Synthetic complete SDS Sodium dodecyl sulphate SDS-PAGE SDS polyacrylamide gel electrophoresis snRNA Small nuclear ribonucleic acid Spl Specificity protein 1 Tafl(3 Template activating factor 1 (3 TR Terminal repeat TRD Transcriptional repression domain Trp Tryptophan Ura Uracil URR Upstream regulatory region YAC Yeast artificial chromosome
XI yEBP2 Yeast EBP2 YPD Yeast extract peptone dextrose I. INTRODUCTION
1.1. The Epstein-Barr Virus
1.1.1. Infection and Associated Diseases
Anthony Epstein and Yvonne Barr first discovered the viral particles of the Epstein-Barr Virus (EBV) in 1964 when they were examining electron micrographs of cells from Burkitt's lymphoma (Epstein et al, 1964). Today, much is known about the promiscuous EBV although more still remains to be unveiled. EBV, also known as human herpesvirus 4 (HHV4), is a gamma-herpesvirus of the genera lymphocryptoviridae (LGV) that is carried by greater than 95% of the adult population. The EBV viral genome exists as linear, double-stranded DNA of-172 Kb in size. Spread via saliva, EBV enters the body through the mucosal epithelium of the oropharynx where it also encounters infiltrating B-cells at the submucosal layer. Upon internalization of the virus into a cell, the two ends of the linear genome which contain terminal repeats ligate and a circular, extrachromosomal plasmid is formed (reviewed in Kieff, 1996; reviewed in Rickinson and Kieff, 1996). This episomal viral DNA is packaged into nucleosomes with a spacing pattern reminiscent of cellular chromatin (Shaw et al., 1979). Within these epithelial cells and a small subset of B-cells, the virus can establish a lytic infection, although EBV infection in B-cells is predominantly latent (see Section 1.1.2.). Primary infection often occurs in early childhood without any remarkable symptoms. However, infection during adolescence or adulthood can result in a self-limiting lymphoproliferative disorder known as infectious mononucleosis (IM), otherwise known as the "kissing disease". Lytic infection involves the expression of approximately 80 viral proteins with roles in replication and viral particle production (Fixman et al, 1992 and references therein). Lytic infection culminates in the shedding of infectious viruses via oral secretions and transmission of the virus to a new host. In immunocompetent individuals, the infection is controlled and the number of infected cells is reduced. However, in some B-cells, the virus escapes surveillance by the immune system and therein establishes a latent infection. Approximately one cell per million of circulating B-cells, which comprises of both resting memory B-cells and cycling blast B-cells, is positive for EBV DNA (Miyashita et al, 1997; Thorley-Lawson et al, 1996). Latently infected cells can be triggered to enter the lytic phase of the viral cell cycle upon expression of
1 either of two EBV immediate-early (IE) genes, BZLF1 and BRLF1, both of which act as potent transactivators of viral lytic gene expression (reviewed in Speck et al, 1997). In cell culture, B-cells undergo transformation and immortalization upon EBV infection, leading to the establishment of lymphoblastoid cell lines (LCLs). Such immortalization is believed to be a normal consequence of EBV latent infection. Although EBV is benign and asymptomatic in the majority of infected individuals, this ability of EBV to exploit B-cells and in rare cases, epithelial cells, can result in several EBV-associated malignancies in both immunocompetent and immunocompromised individuals. Burkitt's lymphoma (BL) and classical Hodgkin lymphoma are examples of B-cell malignancies while nasopharyngeal carcinoma (NPC) is an example of an epithelial cell malignancy. These tumours occur in immunocompetent EBV carriers years after primary infection. In immunocompromised individuals, such as post-transplantation patients or individuals afflicted with acquired immunodeficiency syndrome (AIDS), post-transplantation lymphoproliferative lymphoma and AIDS-associated B-cell lymphomas have been found. The finding that every malignant cell within these tumours contains episomal copies of the EBV genome and viral genes that disrupt normal cell growth suggests that EBV infection may play a causal role in the development of these associated malignancies (reviewed in Kutok and Wang, 2006; Kieff, 1996).
1.1.2. Latent Infection and Viral Gene Expression
EBV infection is usually in a latent state. Latency is characterized by infection without production of new viral particles. During latency, up to 12 different viral genes can be expressed and they include six nuclear proteins [Epstein-Barr virus nuclear antigen 1 (EBNA1), EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA leader protein (LP)], three membrane proteins [latent membrane protein 1 (LMP1), LMP2A, and LMP2B], two small non-polyadenylated Epstein-Barr virus-encoded small RNAs (EBER1 and EBER2), and the transcripts BART/BARFO (reviewed in Rickinson and Kieff, 1996). Depending on the differentiation state of the infected cell, only a subset of these latent genes will be expressed. To date, four different viral gene expression patterns have been described: Latency 0,1, II and III (reviewed in Rickinson and Kieff, 1996). Latency 0 is found in EBV-infected resting memory B-cells where EBERs and possibly LMP2A and BART RNAs are the only genes expressed (Thorley-Lawson et al, 1996; Rowe, 1999; Chen et al, 1999). Latency I, II and III are found in cycling cells
2 (Babcock et ah, 2000; Joseph et ah, 2000; Thorley-Lawson et ah, 1996; Rickinson and Kieff, 2001). Latency I, found in EBV positive Burkitt's lymphoma (BL), is characterized by expression of EBERs and EBNA1 in memory B-cells. Latency II is represented by infected cells found in nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma, and tonsillar memory B-cells. These cells express EBERs, BARTs, EBNA1, LMP1 and LMP2. In addition, NPC cells express the viral BARF1 protein which is secreted from the cells (Seto et ah, 2005). In Latency III, also referred to as the growth program, all 12 latency genes are expressed. Cells in latency III are found in cultured lymphoblastoid cell lines (LCLs), lymphoproliferative disorders in immunocompromised individuals, and in acute infectious mononucleosis (IM). Latent EBV infections persist because the viral genome is able to undergo efficient replication once per cell cycle with the host chromatin followed by stable segregation of the newly-replicated episome to daughter cells during host cell division. Different EBV- immortalized cell lines will harbour different copy numbers of the viral genome, ranging from tens to a few hundred viral plasmids per cell (Sternas et ah, 1990). Remarkably, the viral DNA is maintained in cells at a constant copy number over time. Only two viral components are required for the proper replication and segregation of EBV genomes: the cw-acting origin of latent replication (oriP) (discussed in Section 1.1.3) and the trans-acting factor Epstein-Barr virus nuclear antigen-1 (EBNA1) (discussed in Section 1.1.4).
1.1.3. Viral Origin of Latent Replication (oriP)
During lytic infection, the EBV viral genome is replicated many times in host cells before the viral DNA molecules are packaged into virions that are then released and transmitted to neighbouring host cells. The lytic DNA replication origin, oriLyt, is required to successfully initiate lytic replication (Hammerschmidt and Sugden, 1988). In contrast, replication of the viral genome during latent infection is regulated to only once per cell cycle and requires the viral origin of latent replication, oriP (Adams, 1987; Yates and Guan, 1991). OriP was initially identified by screening EBV DNA fragments for their ability to confer stability to plasmids containing such fragments in human cells that were latently infected with EBV (Yates et ah, 1984). This led to the discovery of an 1800 bp DNA sequence within the EBV episome, which was sufficient for the replication and segregation of plasmids. In fact, plasmids containing oriP can be maintained in human cells at a loss rate of-2-5% per generation
3 even in the absence of selection (Sugden et ah, 1985; Yates et ah, 1984; Yates et ah, 1985). This loss rate is comparable to that obtained for yeast CEN elements, which can undergo autonomous replication and segregation but is nonetheless lost over time as a result of its small plasmid size (Hieter et ah, 1985). Mutational analyses subsequently discovered that oriP was comprised of two functionally-distinct and essential elements separated by approximately 1 Kb of non essential DNA (Reisman et ah, 1985). These elements were the dyad symmetry (DS) element and the family of repeats (FR) (see Figure 1), both of which contain multiple EBNA1 binding sites.
1.1.3. a. DS Element
The DS element is 120 bp in length and contains four EBNA1 recognition sites, each of which is made up of an 18 bp imperfect palindrome sequence to which EBNA1 dimers bind (Ambinder et ah, 1990; Rawlins et ah, 1985). Two of the four EBNA1 recognition sites (EBNA1 sites 3 and 4) form a 65 bp dyad symmetry sequence for which the DS element was named (Rawlins et ah, 1985; Reisman et ah, 1985). The DS element also contains three copies of a 9 bp nonamer sequence, two of which flank each end of the DS element and the third is located between EBNA1 sites 2 and 3. The DS is the site in which replication of the EBV episome and on'P-containing plasmids initiate (Gahn and Schildkraut, 1989; Harrison etah, 1994; Wysokenski and Yates, 1989). Several lines of evidence are provided in support of this hypothesis: First, 2D gel electrophoresis reveals that bi-directional replication initiates at or very near to the DS element (Gahn and Schildkraut, 1989). Second, the human origin recognition complex (ORC), which plays an important role in cellular origin activation, has been found to associate with DS through chromatin immunoprecipitation (ChIP) experiments (Schepers et ah, 2001). Third, the DS component alone, in the absence of FR, can support plasmid replication in a transient transfection assay provided that EBNA1 is also present (Harrison et ah, 1994). It has been shown that the minimal region of DS required for replication is simply two EBNAl-binding sites (either sites 1+2 or sites 3+4) that are properly spaced (Yates et ah, 2000). If the spacing between two EBNAl-binding sites (normally separated by 3 bp) is altered by the addition or deletion of several base pairs, then replication activity is abolished (Yates et ah, 2000; Bashaw and Yates, 2001). While each half of the DS element can act as a minimal core
4 Family of Repeats Dyad Symmetry (FR) (DS)
Figure 1. The EBV latent origin of replication, oriP. The ~ 172 Kb EBV genome encodes the EBV latent origin of replication, oriP, and EBNA1. OriP is composed of a family of repeats (FR) element and a dyad symmetry (DS) element, both of which are separated by ~1 Kb of non essential intervening DNA. FR contains 20 EBNA1-binding sites while DS contains 4 EBNA1- binding sites. Sites 3 and 4 of DS are located within a dyad symmetry sequence for which the element was named and they are indicated by the arrows.
5 replicator, in order to obtain full replication activity, all four sites of the DS element in addition to the three nonamer sequences are required (Koons et al, 2001; Yates et al, 2000).
I.lJ.b.FR Element
The FR element consists of 20 tandem copies of a 30 bp sequence, each of which contains 18 bp of palindromic DNA to which EBNA1 dimers bind and a 12 bp AT-rich sequence (Rawlins et al, 1985; Reisman et al, 1985). EBNA1 binds the FR element with stronger affinity than the DS site (Ambinder et al, 1991; Jones et al, 1989). The slight variations in the 18 bp palindromic sequences found within the FR and DS elements account for the different binding affinities (Ambinder et al, 1990; Summers et al, 1996). The function of FR is distinct from that of the DS element and quite varied. First, FR acts as a transcriptional enhancer when bound by EBNA1 (Reisman and Sugden, 1986; Lupton and Levine, 1985; Reisman et al, 1985). When FR is placed upstream or downstream of a promoter, it can increase transcription of the reporter gene provided that EBNA1 is also present. The FR element is still completely functional as an enhancer element even when up to 13 EBNA1 binding sites are removed (Wysokenski and Yates, 1989), suggesting that the EBNA1 binding sites may play redundant roles. A second function of FR is as a as-acting segregation element even in the absence of DS (Kanda et al, 2001; Kapoor et al, 2001). Plasmids containing FR can be maintained in mitotically active human and budding yeast cells provided that EBNA1 is also present (Krysan et al, 1989; Lupton and Levine, 1985; Simpson et al, 1996; Kapoor et al, 2001). EBNA1 accomplishes this task by interacting with the FR element and tethering the plasmid to condensed human chromosomes as they segregate during mitosis (Harris et al, 1985; Kapoor et al, 2001; Hunger al, 2001; discussed in Section 1.2). The FR element was originally thought to be involved in enhancing DNA replication from the DS element; however, it is now known that this observation is actually the result of increased plasmid retention inside cells as opposed to increased replication efficiency. A third function of the FR is in facilitating the uptake of plasmids into the nucleus (Langle-Rouault et al, 1998). When bound by EBNA1, the FR performs a fourth function as a pause site in both oriP plasmid and EBV episome replication (Gahn and Schildkraut, 1989). Nonetheless, forks have been shown to pass through the FR element and terminate at other sites of the EBV genome (Kirchmaier and Sugden, 1995; Norio et al, 2001).
6 1.1.4. EBNA1 Functions and Domains
In the most commonly studied B95-8 EBV-positive viral strain, the viral protein EBNA1 is 641 amino acids long and 56 kDa in size. Other viral isolates may contain EBNA1 proteins that differ in size due to variations in the length of a glycine-alanine repeat found in the N- terminal half of the protein. EBNA1 plays an important role in latent infection as it is the only trans-acting viral factor that is required for both replication and segregation of or iP plasmids and EBV episomes (Yates et al, 1985). It is also the only EBV protein that is expressed in nearly all latency forms and in all EBV-associated malignancies (see Sections 1.1.1 and 1.1.2). As such, it has been suggested that EBNA1 plays a role in oncogenesis. Thus, gaining insight into how EBNA1 carries out its many functions will prove important for anti-viral drug development.
1.1.4. a. EBNA1 Functional Domains and Structure
EBNA1 is organized into multiple regions that contribute to its various functions. At the N-terminus spanning amino acids 33-53 is a glycine-arginine (Gly-Arg) rich region. This is followed by a large, central glycine-alanine (Gly-Ala) rich repeat spanning amino acids 90-324. A second Gly-Arg region is found in the middle of EBNA1 between amino acids 325-376. The C-terminal portion of EBNA1 contains a nuclear localization signal (NLS; amino acids 379-386), a DNA binding and dimerization region (DBDR; amino acids 459-607) and an acidic tail rich in glutamic acid residues (amino acids 620-641). See Figure 2 for a schematic diagram of the structural domains of EBNA1. The Gly-Ala repeat of EBNA1 varies in size depending on the viral strain. Deletion of this region does not affect the transactivation, replication or segregation functions of EBNA1 (Yates and Camiolo, 1988; Yates et al., 1985). For this reason, the studies performed in our laboratory use a deletion of EBNA1 in the Gly-Ala repeat (EBNA1A101-324) and this version of EBNA1 is considered as wild-type. The role of this large Gly-Ala repeat is in maintaining low EBNA1 turnover rates and evading the host immune response. The Gly-Ala repeat has been known for some time to inhibit efficient presentation of EBNA1 peptides on MHC Class I molecules (Blake et al., 1997), thereby allowing EBNA1 to escape recognition by cytotoxic T cells. Initial studies suggested that proteasome degradation was the main source of peptides for MHC class I presentation and that the presence of the Gly-Ala repeat inhibited EBNA1
7 DNA-binding and Acidic Gly-Arg Gly-Ala Gly-Arg NLS Dimerization tail I * %4 * 4 T2 EBNA1 (B95-8) 1 8 33 5367 90 325 376 386 459 607 641
8 67 325 376 459 607 Replication
8 67 83 325 376 459 Transactivation
8 67 325 376 459 607 1 Segregation 325 376 8 67 Mitotic Chromosome Association
Contribute Required
Figure 2. Schematic diagram of the structural and functional domains of EBNA1. EBNA1 from the B95-8 viral strain is composed of 641 amino acids which make up the following domains: two Gly-Arg regions that span amino acids 33-53 and 325-376, a Gly-Ala repeat found within residues 90-324, a nuclear localization signal (NLS) spanning amino acids 376-386, a C- terminal DNA binding and dimerization region (DBDR) within residues 459-607, and acidic tail between amino acids 620-641. EBNA1 plays many roles inside host cells, including but not limited to replication, transactivation, segregation, and mitotic chromosome association. The domains of EBNA1 that are required (black boxes) or contribute (grey boxes) to these functions are shown.
8 degradation by proteasomes (Levitskaya et al, 1995). More recently, however, it was shown that incomplete translational products, also called DRiPs (defective ribosomal products), are the source of peptides for presentation on MHC Class I molecules instead of peptides generated from the degradation of full-length EBNA1 (Fahraeus, 2005; Tellam et al, 2007). Moreover, it was shown that the Gly-Ala repeat inhibits the formation of DRiPs by slowing EBNA1 translation (Yin et al., 2003), thereby limiting MHC class I presentation. The NLS of EBNA1 allows EBNA1 to gain entry into the nucleus, wherein it resides throughout the cell cycle (Ambinder et al, 1991). The acidic tail of EBNA1 can be removed in the context of full-length EBNA1 without affecting transactivation, replication or segregation (Yates and Camiolo, 1988; Ceccarelli and Frappier, 2000; Polvino-Bodnar and Schaffer, 1992). The role of this acidic tail still remains elusive although it has been suggested that it may affect EBNA1 levels in the cell during viral infection (Ceccarelli and Frappier, 2000). The DBDR of EBNA1 is crucial for all of EBNAl's functions involving oriP, as it is the domain which makes sequence-specific contacts with the 18 bp imperfect palindromic sequences found within the DS and FR elements of oriP (Ambinder et al., 1991; Summers etal, 1996). A prerequisite of DNA binding is EBNA1 dimerization and EBNA1 is able to do so both in solution and when bound to DNA (Ambinder et al, 1991; Frappier and O'Donnell, 1991b). The DBDR alone can serve as a dominant-negative inhibitor of EBNA1 functions by out-competing full-length EBNA1 for binding to oriP plasmids (Kirchmaier and Sugden, 1997). The crystal structure of the DBDR of EBNA1 has been solved both in the presence and absence of the 18 bp EBNA1 recognitions site (Bochkarev et al, 1995; Bochkarev et al, 1996; Bochkarev et al, 1998). This region consists of two domains, termed the core domain (amino acids 504-604) and the flanking domain (amino acids 461-503). The core domain mediates the dimerization of two EBNA1 molecules. It forms an eight-stranded antiparallel p-barrel, comprised of four strands from each monomer and two a-helices per monomer. Mutational analyses later revealed that despite the lack of obvious DNA interactions in the crystal structure, the core domain of EBNA1 does make sequence-specific contacts through one a-helix, referred to as the recognition helix, from each monomer (Cruickshank et al, 2000). The core domain of EBNA1 is strikingly similar to the structure of the DNA binding domain of the E2 viral protein of the bovine papillomavirus although there is a complete lack of sequence homology between the two proteins (Hedge et al., 1992). Moreover, analogous to EBNA1, it is the recognition helix found within the DNA
9 binding domain of the E2 protein that mediates all of the sequence-specific interactions (Hedge et al, 1992). The flanking domain of EBNA1 makes multiple contacts with the DNA and is comprised of an a-helix oriented perpendicular to the DNA and an extended chain that is inserted into the minor groove of the DNA. The core and flanking domains of EBNA1 function together to load the viral protein onto oriP in a sequence-specific manner (Cruickshank et al, 2000). EBNA1 contains a Gly-Arg region at the N-terminus of the protein (amino acids 33-53) and one in the central region (amino acids 325-376). The role of the Gly-Arg region spanning amino acids 33-53 remains unclear. While an EBNA1 mutant that lacks amino acids 8 to 67 has reduced maintenance and transactivation activities (Wu et al, 2002), a smaller deletion encompassing amino acids 33-53 showed maintenance and transactivation activities that were comparable to that of wild-type EBNA1 (Wu et al, 2002). These results suggested that these residues were either not required for these functions or that they played redundant roles with the flanking amino acids within this region. In contrast, the 325-376 Gly-Arg region of EBNA1 makes important contributions in segregation and transactivation, and it has a redundant role in replication along with the 61-83 region of EBNA1 (Wu et al, 2002; discussed in Section I.1.4.b.). This region of EBNA1 has been implicated in protein-protein interactions, RNA binding and nucleolar targeting (Snudden et al, 1994). Moreover, this domain is responsible for the DNA linking and looping phenomena observed for homotypic interactions mediated by EBNA1 bound to oriP (discussed in section I.lAb.L). The central Gly-Arg region of EBNA1 is comprised of an 8-amino acid repeated sequence that occurs six times (Laine and Frappier, 1995). Together, they contain 10 RGG and GRG motifs which are potential sites of arginine methylation, and four serine residues which are potential sites of phosphorylation (Shire et al, 2006). Thus, post-translational modifications of EBNA1 and interactions with cellular proteins mediated through this domain both have important contributions to EBNA1 functions (discussed in greater detail below).
1.1.4. b. EBNA1 Functions
EBNA1 is a complex, multi-functional protein with roles in replication, transactivation, segregation and cellular immortalization. These functions will be discussed in turn below.
10 I.lAb.i. Latent EBV Episome Replication
The initiation of replication of EBV episomes and oriP plasmids involves recruitment of the host cellular replication machinery and changes to DNA structure. An overview of both of these processes will be discussed in this section. EBNA1 is the only viral protein required in the initiation of latent replication (Yates et al, 1985). It has been found that in addition to the essential C-terminal DBDR of EBNA1, the N-terminal half of EBNA1 is also important for replication (see Figure 2) (Yates and Camiolo, 1988; Ceccarelli and Frappier, 2000; Kirchmaier and Sugden, 1997). Mutational analyses revealed that deletion of either amino acids 8-67 or 325-376 had no effect on replication but deleting both regions abrogated the activity (Wu et al, 2002), suggesting that both domains function together to carry out efficient replication. EBNA1 is bound to the DS element at all times of the cell cycle (Hsieh et al, 1993; Niller et al, 1995; Ritzi et al, 2003). However, latent replication initiates only once per S phase, which suggests that EBNA1 binding to the DS element is not sufficient for replication and that EBNA1 lacks any intrinsic enzymatic activity. Moreover, as EBNA1 is the only viral protein required in latent replication, these observations suggest that EBNA1 relies on host cellular proteins to replicate the EBV genome. Indeed, subunits of the origin recognition complex (ORC) and minichromosome maintenance (MCM) complex have been found associated with the DS element (but not the FR element) (Chaudhuri et al, 2001; Dhar et al, 2001; Schepers et al, 2001). It has been previously shown that ORC is recruited by EBNA1 to the DS element (Mien et al, 2004); however, the mechanism in which this occurs is not clear. It is possible that the direct interaction between EBNA1 and ORC (as observed in co-immunoprecipitation assays) places ORC at the DS site (Dhar et al, 2001; Schepers et al, 2001). Alternatively, it is possible that EBNA1-mediated changes in the DNA structure either through nucleosome displacement or DNA bending allows ORC to access the DS element. The presence of ORC at the DS element then recruits MCM (Mendez and Stillman, 2000), which functions as a cellular helicase to separate DNA strands and primes that region for replication (Ishimi, 1997). In keeping with this hypothesis, MCM2, a subunit of the MCM complex, has been found to associate with oriP during Gl and dissociate from it during S phase (Chaudhur et al, 2001). Thus, ORC and MCM have been implicated in the initiation and licensing of DNA replication of the EBV genome. EBNA1 has also been shown to interact with replication protein A (RPA) (Zhang et al, 1998), which coats single-stranded DNA and stabilizes that region so that it may serve as a template for
11 DNA synthesis. The recruitment of RPA to the DS element may be an important early step in the activation of latent origin of replication in EBV. EBNA1 dimers bind to the four recognition sites of the DS element in a cooperative manner (Summers et ah, 1996). The 3 bp interval between adjacent EBNA1 binding sites is crucial for the proper interaction of adjacent EBNA1 dimers and initiation of DNA replication (Bashaw and Yates, 2001; Harrison et al, 1994). The binding of EBNA1 to the DS element has been shown to displace nucleosomes from that site, thereby giving EBNA1 access to the DNA where it can induce structural changes (Avolio-Hunter et ah, 2001). Biochemical studies that include potassium permanganate sensitivity assays show that EBNA1 induces distortions in the DNA upon contact. Specifically, the binding of EBNA1 to two recognition sites within the DS element causes the DNA to bend, resulting in helical distortions and localized regions of overwinding and underwinding that exposes two thymine residues to permanganate oxidation (Frappier and O'Donnell, 1992; Hearing et ah, 1992; Hsieh et ah, 1993; Summers et ah, 1997). Such changes in DNA structure might allow the host replication machinery to gain access to the origin and initiate replication (Frappier and O'Donnell, 1992; Hearing et ah, 1992; Hsieh et ah, 1993; Summers et al., 1997). In addition to cooperative binding of EBNA1 dimers to the DS element, in vitro studies also showed that EBNA1 dimers bound to its recognition sites can undergo homotypic interactions that result in the looping out of DNA if the interaction occurs within an oriP molecule, or in the cross-linking of DNA if the interaction occurs between oriP molecules (Frappier and O'Donnell, 1991; Middleton and Sugden, 1992). Stable homotypic interactions of EBNA1 are mediated predominantly by amino acids 325-376, hence this region is also known as the looping domain. However, amino acids 40-89 have also been shown to mediate weak interactions between EBNA1 monomers (Avolio-Hunter and Frappier, 1998; Frappier et al, 1994; Laine and Frappier, 1995; Mackey et ah, 1995; Mackey and Sugden, 1999). While the significance of DNA looping and linking with respect to EBNA1 function remains unknown, the fact that the regions of EBNA1 that contribute to replication overlap with those required for DNA looping/linking suggests that this activity is important for replication initiation. Indeed, evidence suggests that the ability of EBNA1 to loop DNA stabilizes the EBNA1-DS interaction, allowing for the assembly of a replication complex (Su et ah, 1991; Frappier et ah, 1994).
12 1.1 Ab.ii. Transcriptional Activation
EBNA1 has the ability to both activate and repress transcription of EBV genes; as such, it plays an important role in latent infection. EBNA1-bound FR can activate transcription from the EBV Cp and Wp promoters, which regulate the expression of all EBNAs including EBNA1 during the growth program (Gahn and Sugden, 1995; Sugden and Warren, 1989). Moreover, EBNA1 activates transcription from promoters that regulate the expression of LMPs (Gahn and Sugden, 1995; Sugden and Warren, 1989). Interestingly, EBNA1 can also repress its own expression by binding to two recognition sites upstream of the Qp promoter (Sample et al, 1992; Schaefer et al, 1991). The Qp promoter is responsible for the expression of EBNA1 in latent infection programs other than Latency III (Schaefer et al, 1995). The autoregulatory property of EBNA1 therefore forms an intricate system that allows it to turn on its own transcription when levels are low but also to shut it off when levels are high. Outside the context of the EBV genome, the FR element can be used as a transcriptional enhancer element to increase the expression of reporter genes in the presence of EBNA1 (Reisman and Sugden, 1986; Lupton and Levin, 1985). In mammalian cells, it was shown that the placement of the FR element upstream of the CMV IE or RSV promoter greatly enhanced the expression of the luciferase reporter gene (Langle-Rouault et al, 1998). Moreover, co- transforming an EBNA1-expression vector with a HIS3 reporter gene under the control of the FR element into S. cerevisiae also enhanced transcription of HIS3 (Shire et al, 1999). Only 6-7 EBNA1 binding sites from the FR were required to produce the enhancer effect and this activity could be partially replaced by tandem copies of the DS element (Wysokenski and Yates, 1989). The domains of EBNA1 required for transcription have been mapped to residues 61-83 and 325- 376, in addition to the C-terminal DNA binding and dimerization domain (Wu et al, 2002; Polvino-Bodnar and Schaffer, 1992; see Figure 2). Interestingly, while the 61-83 EBNA1 deletion mutant is active in segregation and replication, it is defective in transcription (Wu et al, 2002), showing for the first time that EBNA1-mediated transcription can be separated from other EBNA1 functions. The mechanism by which EBNA1-mediated transcription occurs still remains largely unclear although several cellular proteins have been implicated in having a role based on their ability to interact with the 325-376 region of EBNA1. These proteins include P32/TAP, Napl, and Tafip, all of which have been shown to interact with residues 325-376 of EBNA1 through
13 affinity chromatography experiments (Holowaty et al, 2003). The reported functions of P32/TAP are wide and varied, ranging from pre-mRNA splicing and transcriptional activation to plasma membrane receptor functions (Wang et al, 1997, and references therein). It has been suggested that P32 may contribute to EBNA1-mediated transactivation (Wang et al, 1997) and EBV latent cycle DNA replication since a fraction of P32 has been found to localize to oriP in vivo (Van Scoy et al, 2000); however, this role has yet to be confirmed. Napl and Tafl(3 are both histone chaperones involved in nucleosome assembly, replication, and transcriptional regulation (Chang et al., 1997; Kawase et al., 1996). The role of Napl in viral transcription has been shown for the bovine papillomavirus where Napl binding to the E2 viral protein enhanced its transcriptional activity (Rehtanz et al, 2004). These results have encouraged our laboratory to investigate the roles of Napl and Tafip in EBNA1-mediated transcription. The 61-83 region of EBNA1 is also required for transactivation. With the modular nature of EBNA1, it is possible that EBNA1 mediates protein-protein interactions at both its N-terminus and central 325-376 region in order to obtain full transactivation activity. To date, no cellular protein has been shown to interact with the 61-83 region of EBNA1.
1.1 Ab.iii. Segregation of the EBV Episome and EBV-Based Plasmids
The ability of EBV to persist in latently infected cells is attributed not only to the ability of EBNA1 to replicate the EBV genome in cells, but also to the efficient ability of EBNA1 to segregate the EBV episome stably between dividing daughter cells. Since the EBV genome does not contain a centromeric sequence, the mechanism of episome partitioning is different from that of host chromosome segregation. The mitotic tethering model has been proposed to explain how the EBV episome is transmitted stably in host cells. In brief, the model suggests that episome- bound EBNA1 tethers to sister chromatids on host mitotic chromosomes and thereby places the EBV genome at a location which ensures its faithful segregation to daughter cells at the end of mitosis. This model will be discussed in greater detail in Section 1.2. In this section, the viral components and EBNA1 domains required for segregation will be discussed. Episome maintenance requires only the FR element of oriP and EBNA1 in trans (Krysan et al, 1989). This conclusion was drawn from several independent experiments which showed that the presence of FR and EBNAl can confer maintenance stability to exogenous plasmid
14 constructs that contain heterologous origin of replication sequences. This occurs both in human cells (Krysan et ah, 1989; Simpson et ah, 1996) as well as in budding yeast (Kapoor et ah, 2001). The modularity of EBNA1 allows its N-terminus to attach to mitotic chromosomes while its C-terminus binds the EBV episome (Marechal et ah, 1999; Shire et ah, 1999; Wu et ah, 2000; Hung et ah, 2001; Wu et ah, 2002). Deletions in the context of full-length EBNA1 have revealed that the 325-376 region is critical for chromosome attachment and segregation function (see Figure 2). Indeed, the EBNA1A325-376 mutant was disrupted in plasmid maintenance ability and mitotic chromosome interactions (Shire et ah, 1999; Wu et ah, 2000; Wu et ah, 2002). Furthermore, mutations of serine residues within this region indicated that phosphorylation of these residues may enhance segregation although other residues within this region may also play a role (Shire et ah, 2006). It is important to point out that while such a mutation affected segregation, replication was still functional (Yates and Camiolo, 1988; Shire et ah, 1999). Therefore, EBNAl's segregation function can be separated from its replication function. An EBNA1 mutant lacking amino acids 8-67 showed a four-fold decrease in oriP plasmid maintenance ability compared to wild-type EBNA1 (Wu et ah, 2002). In keeping with this observation, EBNA1A8-67 showed slightly more diffused staining on mitotic chromosomes compared to wild-type EBNA1 (Wu et ah, 2002). This is consistent with reports by others that the N-terminal sequences might contribute to chromosome binding. Marechal et al. (1999) showed that EBNA1 peptides spanning amino acids 8-54, 72-84 and 328-365, each fused to green fluorescent protein (GFP), were able to bind mitotic chromosomes on their own. Moreover, in a similar experiment, Hung et al. (2001) showed that GFP-tagged EBNA1 fragments spanning amino acids 1-89 and 323-386 were each able to associate diffusely with mitotic chromosomes, suggesting that each fragment associated weakly with cellular DNA. However, strong chromosome association indicated by intense staining of mitotic DNA was seen only when an EBNA1 fragment spanning both regions were used. Initially, it was suggested that the contribution of residues 8-67 to chromosome attachment was due to the Gly-Arg repeat at amino acids 33-53. Subsequently, it was shown that this sequence can be deleted from EBNA1 without affecting plasmid maintenance, suggesting that this region of EBNA1 either has no role in segregation or that other residues within the 8-67 domain have a redundant contribution (Wu etah, 2002).
15 I.lAb.iv. Cell Immortalization
In vitro, primary human B-cells that are infected by EBV acquire the characteristic of unlimited cell proliferation, leading to the establishment of lymphoblatoid cell lines (LCLs). In vivo, latently infected cells may develop EBV-associated malignancies (see Section 1.1.1.). Several key observations lead to the hypothesis that EBNA1 plays an important role in the transformation and immortalization of these cells. First, EBNA1 is the only viral protein expressed in all EBV-related tumours (reviewed in Kieff and Rickinson, 2001). Second, expression of EBNA1 in transgenic C57BL/6 mouse lines led to increased cell proliferation and a high incidence for B-cell lymphoma development (Wilson et al, 1996). It is worth mentioning that the contribution of EBNA1 to tumour formation has been questioned by others (Kang et al, 2005; Kang et al, 2008). However, a further analysis on this subject is beyond the scope of this thesis. A third observation implicating EBNA1 as an oncogenic factor is that down-regulation of EBNA1 by RNA interference led to decreased cell proliferation in Raji Burkitt's lymphoma and nasopharyngeal carcinoma cells (Hong et al, 2006; Yin and Flemington, 2006). Fourth, EBNA1 expression in U20S cells protects them from apoptosis by reducing the accumulation of p53 in response to DNA damage (Saridakis et al, 2006). The mechanism by which EBNA1 contributes to cell immortalization or transformation is unclear but it may involve several mechanisms. Such mechanisms may range from altering cellular gene transcription levels to sequestration of key cell-cycle regulators such that they can no longer perform their normal functions. Our laboratory has shown that EBNA1 is able to interact with and thereby sequester herpes-associated ubiquitin specific protease (HAUSP), also known as ubiquitin specific protease 7 (USP7) (Howlowaty et al, 2003a; 2003b). HAUSP/USP7 is a protein that binds to, deubiquitinates, and subsequently stabilizes the oncogenic protein p53 (Li et al, 2002; Cummins et al, 2004; Cummins and Vogelstein, 2004). As such, HAUSP/USP7 is an important regulator of normal cellular apoptosis. EBNA1 can out- compete p53 for binding HAUSP/USP7 and in doing so, p53 is destabilized (Saridakis et al, 2006). As a result, cells undergo decreased p53-dependent apoptosis upon induced DNA damage and show increased cell survival (Saridakis et al, 2006).
16 1.2. Mechanism of EBV Segregation
The mitotic tethering or "hitchhiking" model of EBV is currently the favoured model to explain how the viral genome is distributed to daughter cells at the end of mitosis. In this section, the mitotic tethering model will be discussed in greater detail with an emphasis on the role of human EBNA1-binding protein 2 (hEBP2) in mediating EBNA1-dependent partitioning of EBV- based plasmids. Furthermore, a brief overview of how newly replicated EBV episomes are symmetrically localized on sister chromatids of mitotic chromosomes will also be discussed. The emphasis in this latter section is on emerging data which suggest that replication and mitotic tethering oforiP plasmids are coupled processes that together ensure faithful segregation of EBV episomes.
1.2.1. The Mitotic Tethering or "Hitchhiking" Model of EBV
The first clue that EBV episomes piggyback or hitchhike onto cellular chromosomes in order to ensure their proper partitioning and stable transmission to daughter cells came from immunofluorescence microscopy experiments which showed that EBNA1 localized to mitotic chromosomes of Raji cells (Grogan et ah, 1983). Subsequently, fluorescence in situ hybridization (FISH) experiments showed that EBV episomes also associated with metaphase chromosomes of Burkitt's lymphoma (BL) cells (Harris et ah, 1985). Moreover, this linkage between the EBV episome and DNA was non-covalent since increasing the temperature of chromosome denaturation in the hybridization protocol removed the vast majority of EBV episomes from metaphase chromosomes (Harris et ah, 1985). The mitotic tethering model soon became the preferred dogma to explain the faithful segregation of EBV episomes. Several lines of evidence were later provided to support this model and shed light into the mechanism by which this intricate process occurred. First, others were able to show that EBNA1 bound to mitotic chromosomes (Petti et ah, 1990). Second, oriP plasmids were able to tether to cellular chromosomes only in the presence of functional EBNA1 (Simpson et ah, 1996). Third, EBNA1- oriP plasmids co-localized on condensed mitotic DNA (Kanda et ah, 2001). Fourth, the FR element of oriP was shown to be sufficient for plasmid attachment to chromosomes and for its faithful segregation to daughter cells even in the absence of the DS element (Krysan et ah, 1989; Kanda et ah, 2001). Fifth, an EBNA1 mutant lacking amino acids 325-376 that was defective in
17 segregation was also defective in chromosome attachment (Hung et al, 2001; Shire et al, 1999; Wu et al, 2000). Sixth, to confirm the contention that plasmid maintenance is achieved by EBNA1 attachment to mitotic chromosomes, Hung et al. found that substituting the mitotic chromosome association portion of EBNA1 with chromosome-binding sequences of HMG-1 (high-mobility group-1) or histone HI was sufficient for EBNA1 chromosome attachment and oriP plasmid maintenance (Hung et al., 2001). EBNA1 attachment to mitotic chromosomes is most likely mediated by interaction with a chromosome-bound cellular protein. The rationale behind this hypothesis is that the chromosome attachment domain of EBNA1 is the 325-376 Gly-Arg rich region (Hung et al, 2001; Shire et al, 1999; Wu et al, 2000) and Gly-Arg rich motifs are known protein-protein interaction modules. Moreover, other viral proteins that mediate segregation interact with mitotic chromosomes through cellular proteins (see Section 1.3.), suggesting that this mechanism of episome partitioning could be conserved. In support of this hypothesis, our laboratory discovered that hEBP2 plays an important role in mediating EBNA1 -association with mitotic chromosomes. This will be discussed in greater detail in the next section. It is worth noting at this point that one study suggests that EBNA1 may be able to associate directly with DNA through the Gly-Arg repeats found within amino acids 33 to 89 and amino acids 328 to 378, since they resemble AT-hooks (Sears et al, 2004). However, deletion of the N-terminal putative AT-hook has no effect on EBNA1 segregation function (Wu et al, 2002). Moreover, the central Gly-Arg repeat is known to bind several cellular proteins and therefore it is not clear if this region is very available to bind DNA in the context of the folded EBNA1 protein. Nevertheless, the finding that EBNA1 does not associate with yeast chromosomes unless supplied with hEBP2 (Kapoor et al, 2001; discussed below) is not consistent with the AT-hook model. However it is still possible that direct DNA interaction could partially contribute to chromosome attachment.
1.2.2. The Role ofhEBP2 in EBNA1-MediatedSegregation of EBV-Based Plasmids
EBNA1 mediates segregation of the EBV episome by attaching to the FR element of or iP and tethering the viral DNA to mitotic chromosomes. EBNA1 is thought to accomplish this task by interacting with a chromosome-bound cellular protein. Our lab identified one such cellular protein as human EBNA1-binding protein 2 (hEBP2). Extensive work in both budding yeast and
18 human cells has revealed the importance of hEBP2 in EBV episome partitioning and viral persistence.
1.2.2.a. Properties and Cellular Function of EBP 2
hEBP2 was first identified by monoclonal antibodies generated from immunizing mouse cells with the nucleolus of HeLa cells (Chatterjee et al, 1987). This monoclonal antibody
recognized a specific protein band of Mr 40,000 in nucleolar lysates; thus, hEBP2 was actually first coined the name nucleolar protein p40. The objective of the initial study was to identify differences in the nucleolus of resting and dividing tumour cells since it was known that the nucleoli of tumour cells were hyperactive compared to normal cells (Busch and Smetana, 1970). In this study, it was discovered that while hEBP2 was found in the nucleolus of dividing tumour cells (i.e. HeLa), it was absent in resting, normal human tissues such as liver and kidney cells, suggesting that hEBP2 had a role in cell proliferation. It is now known that hEBP2 is a 35-kDa protein consisting of 306-amino acids which together make up three domains: an N-terminus, a central domain, and a C-terminus (see Figure 3). Through data bank searches, homologues of hEBP2 can be found in Saccharomyces cerevisiae (budding yeast), Schizosaccharomycespom.be (fission yeast), and Caenorhabditis elegans (nematode worm). Interestingly, a protein sequence alignment of EBP2 from these four organisms reveal highly conserved middle and C-terminal domains but divergent N-terminal portions (Shire et al, 1999). Indeed, the C-termini of all four proteins share a conserved 200- 300 amino acid block of residues with at least 38% sequence identity (Huber et al, 2000). Furthermore, while the human and S. pombe EBP2 proteins are similar in length, that of S. cerevisiae and C. elegans are longer due to larger extensions at the N-termini. A secondary structure prediction program (PHDsec from EMBL, Heidelberg, Germany) reveal that hEBP2 has 47% a-helical character and that the middle region of hEBP2 (approximately amino acids 100-208) consists of a coiled-coil domain which is predicted to participate in protein-protein interactions (Shire et al, 1997). Like hEBP2, the yeast homologue of EBP2 is also found in the nucleolus (Huber et al, 2000; Tsujii et al, 2000). yEBP2 is 427 amino acids long and it is an essential gene in yeast. Viability in the EBP2 yeast deletion strain (ebp2::HIS3) can be restored when the strain is transformed with a plasmid encoding yEBP2 or even with a fragment of yEBP2 that lacks the
19 Coiled-coil domain hEBP2 N 220 306
v. Y Y Mitotic EBNA1 Chromosome Association Association
Figure 3. Schematic diagram of the structural and functional domains of hEBP2. hEBP2 is composed of 306 amino acids which can be divided into three domains: an N-terminus, a coiled- coil middle domain, and a C-terminus. The coiled-coil middle domain governs hEBP2 association with mitotic chromosomes while the C-terminus binds EBNA1.
20 first 178 amino acids of the protein, suggesting that the C-terminus of the protein is critical for its activity while the N-terminus is dispensable (Huber et al, 2000). A yeast strain containing a temperature-sensitive allele of EBP 2 showed reduced ribosome content compared to the wild- type parent strain (Huber et al, 2000; Tsujii et al, 2000). Subsequently, it was shown that this was due to defects in the processing of the 27SA into the 27SB precursor rRNA, which under normal conditions would be further processed into the 5.8S and 25S rRNAs found in ribosomes. Since hEBP2 and yEBP2 are highly homologous and both proteins are found in the nucleolus, it is generally assumed that hEBP2 is also involved in rRNA processing in human cells.
1.2.2.b. Interaction Between EBNA1 and human EBNA1-Binding Protein 2 (hEBP2)
The interaction between EBNA1 and hEBP2 was discovered through a yeast two-hybrid screen from a human B-lymphoma cDNA library (Shire et al, 1999). This interaction was also observed by co-immunoprecipitation (co-IP) experiments using baculovirus-produced EBNA1 and hEBP2 in insect cells. By conducting a yeast one-hybrid experiment, it was discovered that hEBP2 fused to a GAL4 activation domain can successfully associate with FR-bound EBNA1 to activate the HIS3 reporter gene (Shire et al, 1999). Thus, hEBP2 can interact with both DNA- bound forms and free forms of EBNA1. It is not yet known whether the interaction between EBNA1 and hEBP2 is direct or indirect. However, since the interaction has been observed in both yeast and insect cells, it is likely that their interaction is direct and not mediated by another protein. Importantly, it is unlikely that EBNA1 and hEBP2 associate through nucleic acids since treating insect cell lysates containing baculovirus-produced proteins with RNase or DNasel did not abrogate their interaction (Shire et al, 1999). One of the first clues that prompted the investigation of hEBP2 in EBNA1-mediated segregation came from studies mapping the hEBP2-interaction domain of EBNA1. Using yeast two-hybrid assays and co-IP experiments where hEBP2 and a series of EBNA1 truncation mutants were expressed in insect cells, our lab discovered that hEBP2 interacted with the 325- 376 Gly-Arg region of EBNA1 (Shire el al, 1999). As described previously (Section I.lAb.ii.), this region of EBNA1 has been shown to be required in the maintenance of oriP plasmids (Shire et al, 1999). Interestingly, smaller deletions within this region (EBNA1A367-376 and EBNA1A 356-362) can still bind hEBP2 and support oriP plasmid maintenance (Shire et al, 1999). Thus, the correlation between the EBNA1 residues required for hEBP2 binding and those required for
21 plasmid maintenance implicated hEBP2 as an important player in EBV segregation. Subsequent work in both budding yeast (see Section 1.2.2.c.) and human cells (see Section 1.2.2.d.) done in the laboratory would confirm the importance of the EBNAl-hEBP2 interaction in the partitioning of EBV episomes during cell division. To map the EBNA1-interaction domains of hEBP2, yeast two-hybrid experiments were conducted (Kapoor et al, 2001). EBNA1 fused to the GAL4 DNA-binding domain was tested with several hEBP2 fragments fused to the GAL4 activation domain. It was found that EBNA1 bound the C-terminal domain of hEBP2 spanning amino acids 220-306 (see Figure 3). In contrast, the N-terminal domain and the central coiled-coil domain of hEBP2 were incapable of activating the HIS3 and LACZ reporter genes. The yeast homologue of EBP2 was also tested for its ability to interact with EBNA1 and the results suggest that yEBP2 is sufficiently different from hEBP2 since yEBP2 and EBNA1 do not interact (Kapoor et al, 2001).
1.2.2. c. hEBP2 Reconstitutes EBNA1-Mediated Plasmid Maintenance in Budding Yeast
The study of EBNA1-mediated segregation of or/P-containing plasmids in human cells is complicated by the fact that maintenance of this plasmid depends on the ability of EBNA1 to both replicate and faithfully segregate the plasmid to daughter cells after each cell division. As it is difficult to separate the replication and segregation functions of EBNA1 in human cells, long- term plasmid maintenance assays must be accompanied by transient plasmid replication assays to ensure that any loss of the segregation test plasmid in cells is not due to the inability of EBNA1 to mediate its replication but due to a deficiency in its maintenance. In order to overcome this obstacle, our lab developed a yeast plasmid loss assay, which measures EBNA1-mediated segregation in the absence of EBNA1 -mediated replication. The yeast plasmid loss assay utilizes Saccharomyces cerevisiae and an unstable yeast replicating plasmid containing an origin of replication (ARS) but lacking a centromeric element. The EBV FR element was inserted into this plasmid to determine if it would support partitioning in the presence of EBNA1 (Kapoor et al, 2001). EBNA1 alone did not support partitioning in this system but, since hEBP2 was suggested to play a role in segregation, it was tested for its ability to rescue EBNA1-mediated partitioning. Indeed, the presence of hEBP2 reconstituted the segregation of the FR-containing plasmid in an EBNA1-dependent fashion (Kapoor et al, 2001). While the C-terminal domain of hEBP2 (amino acids 220-306) was found to be the EBNA1-
22 binding domain, this fragment was insufficient to support maintenance of the segregation test plasmid (Kapoor et al, 2001). Maintenance was achieved if a fragment of hEBP2 containing the central coiled-coil domain and the C-terminal domain (amino acids 95-306) was used in the presence of EBNA1, suggesting that while the C-terminus of hEBP2 interacts with EBNA1, it is the middle region which associates mitotic chromosomes (see Figure 3) (Kapoor et al, 2001). In support of this hypothesis, it was found that a fusion protein containing the middle region of hEBP2 (amino acids 220-306), the nuclear localization signal of EBNA1 (amino acids 376-386), and the DNA-binding and dimerization domain of EBNA1 (amino acids 452-641) can confer maintenance stability to the FR-containing plasmid (Kapoor and Frappier, 2003). Subsequent plasmid loss assay studies also revealed that an EBNA1 mutant lacking amino acids 325-376 could not mediate plasmid maintenance in yeast (Kapoor et al, 2001), consistent with the idea that the interaction between hEBP2 and EBNA1 is required for stable plasmid partitioning in cells. Moreover, an EBNA1 mutant lacking amino acids 8-67 showed a three-fold reduction in plasmid maintenance ability compared to wild-type EBNA1 (Wu et al, 2002). These results correlate well with studies performed in human cells which showed that deletion of amino acids 8-67 of EBNA1 reduced or/P-containing plasmid maintenance by four-fold compared to wild- type EBNA1 (Wu et al, 2002; see Section I.lAb.ii). It is thought that residues 8-67 stabilize EBNA1 association with hEBP2 on host mitotic chromosomes, thereby allowing for optimal segregation activity. In keeping with this hypothesis, our laboratory has shown that EBNA1A8- 67 binds weakly to hEBP2 compared to wild-type EBNA1 (Kapoor and Frappier, unpublished data). Taken together, these results suggest that although not required for segregation, amino acids 8-67 of EBNA1 do contribute to this function. Indirect immunofluorescence studies on yeast metaphase spreads revealed that both EBNA1 and hEBP2 showed significant amount of overlap on mitotic chromosomes with a staining pattern similar to that of DAPI (Kapoor and Frappier, 2003). While EBNA1 binding to mitotic chromosomes required hEBP2, hEBP2 binding to DNA occurred both in the presence and absence of EBNA1. Moreover, an hEBP2 mutant that lacked the central coiled-coil domain essential for mitotic chromosome association failed to tether EBNA1 to mitotic chromosomes (Kapoor and Frappier, 2003). These results provided strong evidence that FR plasmid maintenance in yeast cells required EBNA1 binding to chromosome-bound hEBP2, thereby allowing for proper partitioning of the FR plasmid to daughter cells.
23 Surprisingly, while yeast EBP2 is highly homologous to human EBP2 (see Section 1.2.2.a), it cannot support EBV-based plasmid maintenance even when yeast cells were provided with yEBP2 exogenously on a plasmid (Kapoor and Frappier, 2003). An investigation into yEBP2 revealed two key reasons to explain why the human and yeast versions of EBP2 function differently in EBNA1-mediated partitioning in budding yeast (Kapoor and Frappier, 2003). First, yEBP2 does not interact with EBNA1 in a yeast two-hybrid system. Second, yEBP2 is confined to the nucleolar region of DNA throughout mitosis while the human form associates more extensively with mitotic chromosomes. Even when the C-terminal domain of yEBP2 was "swapped" for that of hEBP2 (the EBNA1-binding region spanning amino acids 220-306), plasmid maintenance was still not observed likely because, unlike hEBP2, yEBP2 does not re- localize from the nucleolus to form more extensive contacts with mitotic DNA. Thus, yEBP2 is defective in segregation because it cannot bind EBNA1 or associate with mitotic chromosomes.
1.2.2. d. hEBP2 Plays an Important Role in EBNA1-Mediated Segregation in Human Cells
Human EBP2 also plays an important role in EBNA1-mediated segregation in human cells. Indeed, several lines of evidence suggest that hEBP2 functions similarly in yeast cells as it does in human cells to tether EBNA1 and or/'P-containing plasmids to mitotic chromosomes. First, immunofluorescence studies on metaphase spreads done in our laboratory showed that both GFP-tagged and endogenous hEBP2 can associate with mitotic chromosomes and a significant amount of overlap with EBNA1 is observed (Kapoor and Frappier, 2003; Kapoor et ah, 2005). Second, hEBP2 and EBNA1 are found in the chromosome-bound fraction in biochemical fractionation experiments (Kapoor et al, 2005). This is true for cell lines wherein EBNA1 is exogenously expressed on a plasmid as well as for EBV-positive cell lines. However, upon silencing hEBP2, approximately 51% to 75% of EBNA1 (depending on the cell line examined) is released from the chromosome and enters the soluble fraction (Kapoor et al, 2005). Third, concomitant immunofluorescence studies done in hEBP2-silenced cells showed the dissociation of EBNA1 from mitotic chromosomes, with residual EBNA1 staining the outer edges of the chromosome not stained by DAPI. Fourth, silencing hEBP2 also disrupted the attachment of onP-containing plasmids to mitotic chromosomes (Kapoor et al, 2005). Fifth, in an independent study, it was shown that maintenance of an EBV-derived episomal vector in the murine Sp2/0 cell line required both EBNA1 and exogenous expression of hEBP2 (Habel et
24 ah, 2004). These studies provide strong support for the importance of hEBP2 in mediating EBNA1 and EBV-based plasmid attachment to mitotic chromosomes, thereby allowing for faithful segregation of viral episomes and persistence of EBV in mammalian host cells.
1.2.2.e. EBP2 is Regulated by Aurora Family Kinases
The mechanism by which hEBP2 attaches to mitotic chromosomes is still not well understood. Currently, we favour the model where hEBP2 associates with a chromosome-bound protein as opposed to interacting directly with DNA for two reasons: First, hEBP2 associates with mitotic chromosomes through its coiled-coil domain (Kapoor and Frappier, 2003), which is a known protein interaction module. Second, hEBP2 is able to make extensive contacts with the highly condensed and mostly inaccessible chromosome during mitosis. In order to elucidate some of the possible protein players involved in hEBP2 binding to mitotic chromosomes, our laboratory used a series of ts yeast strains that have compromised mitotic chromosome structure and function to examine the effect this has on hEBP2 attachment to yeast DNA during mitosis (Kapoor et ah, 2005). These studies showed that neither proper sister chromatid cohesion nor chromosome condensation were important for hEBP2 localization to DNA during mitosis. However, loss of Ipll kinase function in yeast or down-regulation of the equivalent Aurora B kinase homologue in human cells resulted in the dissociation of hEBP2 from mitotic chromosomes. Ipll and Aurora B are protein kinases known to phosphorylate histone H3, kinetochore components, and subunits of the condensin and cohesion complexes. As such, they play important roles in chromosome condensation, segregation and cytokinesis (Carmena and Earnshaw, 2003; Biggins et ah, 1999; Cheeseman et ah, 2002; Hsu et ah, 2000; Lavoie et ah, 2004). Since hEBP2 lacks the consensus phosphorylation site preferred by Ipll, it is thought that the observed dissociation of hEBP2 from yeast mitotic chromosomes upon loss of Ipll function is due to loss of phosphorylation of a chromosomal protein to which hEBP2 binds as opposed to loss of hEBP2 phosphorylation by Ipll kinase. The identity of this phosphorylatable chromosomal protein remains unknown.
25 1.2.2.f. The Role ofhEBP2 in the Mitotic Tethering Model ofEBV Our lab has provided strong evidence supporting the role of hEBP2 in the mitotic tethering model of EBV. See Figure 4 for a schematic of the segregation model of EBV. In summary, the DBDR of EBNA1 binds the FR element oforiP and the viral episome or EBV- based plasmid is then tethered to mitotic chromosomes through association with hEBP2. The 325-376 Gly-Arg rich region of EBNA1 binds the C-terminus of hEBP2 whose coiled-coil middle domain spanning amino acids 95-220 is responsible for attaching the complex to mitotic DNA. The 8-67 region of EBNA1 also contributes to hEBP2-mediated segregation in yeast, presumably by stabilizing the interaction with hEBP2. It is hypothesized that hEBP2 localizes to mitotic chromosomes by associating with another chromosome-bound protein that is regulated by Aurora B protein kinase (protein X). Tethering of this complex to mitotic chromosomes allows for viral persistence since the EBV genome is placed at a location that ensures its proper segregation along with sister chromatids at the end of mitosis.
1.2.3. Even Distribution of EBV Episomes on Sister Chromatids Leads to Equal Partitioning
It is widely believed that EBV segregation must involve a mechanism to ensure that the viral episomes are equally distributed on the paired sister chromatids during mitosis such that they will be equally partitioned between daughter cells after cell division. Indeed, latently infected cells have the ability to maintain from tens to hundreds of EBV episomes per cell at constant copy number over many generations (reviewed in Kieff, 1996; Sternas et al., 1990). Burkitt's lymphoma cells carry on average from less than 1 to approximately 10 copies of EBV episomes per cell. If EBV had a stochastic mechanism for partitioning low copy-number viral genomes, it would be difficult to envision how cells are able to remain latently infected even after many years. In the past, FISH experiments suggested that oriP associated randomly with chromosomes and was not targeted to specific chromosomal regions such as centromeres or telomeres (Harris et al, 1985). More recently, new approaches revealed that while episomes are not targeted to specific chromosomal regions, their association with DNA may not be so random (Delecluse et al, 1993; Simpson et al, 1996; Kanda et al, 2001; 2007; Nanbo et al, 2007). Delecluse et al. (1993) showed that in Burkitt's lymphoma cells, FISH signals targeting the EBV episome were symmetrically localized on sister chromatids in mitosis. Simpson et al. (1996) also observed the pairing of plasmid dots on metaphase chromosomes when they
26 Mitotic Chromosomes
Figure 4. hEBP2 and EBNA1-mediated mitotic tethering model of EBV. EBNA1 binds to the FR element of the EBV genome through the C-terminal DNA binding and dimerization domain spanning amino acids 452-607. EBNA1 is tethered to host mitotic chromosomes through association with hEBP2. EBNA1 amino acids 325-376 are required for binding hEBP2 and residues 8-67 may contribute to the interaction. The region of hEBP2 involved in EBNA1 binding lies within amino acids 220-306 and residues 95-220 are responsible for mitotic chromosome tethering. hEBP2 is thought to attach to mitotic DNA via a chromosome-bound cellular protein (protein X) whose identity is not known. Nevertheless, protein X is possibly a substrate of Aurora B kinase and is regulated by phosphorylation (Pn). The placement of EBV genomes on mitotic chromosomes ensures that they will be faithfully partitioned to daughter cells during host cell division.
27 transfected human kidney 293 cells stably expressing EBNA1 with OriPYAC, a yeast artificial chromosome containing the oriP sequence. More recently, two independent groups of investigators have shown that oriP plasmids localize symmetrically on sister chromatids after replication in S phase and remain in this manner during G2 (Kanda et al, 2001; 2007; Nanbo et al, 2007). This symmetrical localization of oriP plasmids likely represents catenated plasmids that are formed at the end of replicating the circular DNA molecule prior to their separation by topoisomerase II. Indeed, using a topoisomerase II inhibitor led to an increase in the frequency of paired plasmids present in each cell, presumably due to an increase in catenated plasmids that could not be separated (Kanda et al, 2007). Moreover, in cells expressing low EBNA1 levels, a co-localization of paired EBNA1 dots where there is a paired oriP plasmid can also be seen (Kanda et al, 2007). The authors also noted that pairing of plasmids can be seen during mitosis after which each daughter cell receives the same number of EBV episomes or EBV-based plasmids. Based on these observations, the authors proposed that there is a link between the pairing of replicated plasmids through catenation and equal partitioning. Therefore, it is through this non-stochastic coupling of synthesis and segregation that EBV episomes are'maintained stably in latently-infected cells. These studies beg the following questions: can EBNA1 bind interphase chromatin and how does the role of hEBP2 in EBNA1-mediated segregation fit into this model? While it has been conclusively shown that EBNA1 binds mitotic chromosomes and is nuclear during interphase, whether EBNA1 binds interphase chromatin remains unclear. Several immunofluorescence and biochemical studies have shown that EBNA1 localizes to prematurely condensed chromatin and fractionates with interphase chromatin (Ito et al, 2002; Kanda et al, 2001; Kanda et al, 2007; Nanbo et al, 2007). However, others have shown that EBNA1 is found in the soluble fraction in biochemical fractionation experiments (Daikoku et al, 2004). Nevertheless, if EBNA1 does interact with interphase chromatin, it is likely not mediated by hEBP2 since hEBP2 is confined to the nucleolus during interphase (Chatterjee et al, 1987). At this time, one can only speculate that the DNA association mechanisms of EBNA1 differ during interphase and mitosis. It is possible that EBNA1 binds directly to interphase chromatin through its AT hooks as predicted by other investigators (Sears et al, 2004). However, during mitosis, EBNA1 association with chromosomes probably involves a combination of methods that includes binding to hEBP2.
28 1.3. Mechanism of Segregation of Other Viruses
The mitotic tethering model for viral episome partitioning is not unique to only EBV but is also used by other viruses whose genome exists as low copy number, extrachromosomal, double-stranded, circular DNA molecules. Like EBV, stable genome segregation only requires two viral factors: a cw-acting factor and a trans-acting factor, of which the latter binds to the cis element and tethers the episome to host mitotic chromosomes. Thus, there appears to be a conserved mechanism of chromosomal association that is used by these viruses to ensure stable persistence in latently infected cells. In this section, I will discuss the segregation mechanism of the bovine papillomavirus type-1 (BPV1), Kaposi's sarcoma-associated herpesvirus (KSHV), and herpesvirus saimiri (HVS). See Figure 5 for a schematic of the proposed mitotic tethering models for these viruses.
1.3.1. Bovine Papillomavirus Type-1 (BPV1)
Papillomaviruses (PVs) are small double-stranded DNA viruses of-8000 bp in size that are found in a wide range of mammals. PVs have a specific tropism for either squamous epithelial cells (all human PVs) or fibroblasts (bovine PV). In humans, they can cause benign lesions or warts in the skin and genital areas; however, they have also been intricately associated with the development of malignant carcinomas. Indeed, several human papillomavirus (HPV) types, such as HPV 16, 18, 31 and 45, are considered as "high-risk" because they have been associated with some human cancers, particularly cervical cancer. All PV types can establish a latent infection by maintaining their viral genome as low-copy number episomes (reviewed in Howley, 1996). Like EBV, this is established by efficient replication of the viral genome followed by its faithful segregation. Bovine papillomavirus type-1 (BPV1) has been the prototypic virus that has served as a model for studies of PV episome maintenance. As such, this will be the virus discussed here. Two viral factors are necessary and sufficient for the replication of the BPV1 genome: El and E2 (Ustav and Stenlund, 1991). Moreover, the minimal origin (MO) of replication is also required in cis (Ustav et ah, 1991). The minimal origin of replication is located near the ~1 kb non-coding upstream regulatory region (URR) and contains a binding element for El that is flanked on one side by one binding site for E2 and on the other side by an AT-rich region (Ustav
29 Bovine Papillomavirus Kaposi's Sarcoma- Herpesvirus Type-1 Associated Herpesvirus Saimiri (BPV1) (KSHV) (HVS)
Figure 5. The proposed mitotic tethering models of BVP1, KSHV and HVS. The mitotic tethering model is the favoured dogma to explain the segregation of extrachromsomal viral genomes such as BPV1 (A), KSHV (B) and HVS (C). In this model, a latent viral protein binds to its cognate site in the viral genome and tethers it to mitotic chromosomes to ensure its stable partitioning to daughter cells during host cell division. (A) The E2 viral protein of BPV1 is thought to tether MME-containing plasmids to host mitotic chromosomes through association with the cellular proteins ChlRl and Brd4. (B) Several cellular proteins have been implicated in tethering KSHV LANA1 bound to the TR element of the viral genome to mitotic chromosomes, including MeCP2, Brd2, Brd4 and DEK. No functional studies have been done to confirm their roles in KSHV segregation although all proteins have been shown to bind to and co-localize with LANA1 on mitotic DNA. (C) HVS ORF73 associates with TR-containing plasmids and has been proposed to be tethered to condensed chromosomes via an interaction with MeCP2.
30 et al, 1991). El is a virally encoded helicase that is important for initiation and elongation of replication (Ustav and Stenlund, 1991; Ustav et al, 1991). It has been shown to interact with DNA polymerase a-primase and to replication protein A (Han et al, 1999; Melendy et al, 1995; Park et al, 1996), which may lead to assembly of a replication complex at the BPV1 origin. E2 is a DNA-binding transcription factor (Spalholz et al, 1985) that regulates gene expression from the viral promoter (Romanczuk et al, 1990; Thierry and Yaniv, 1987). It also forms a complex with El and mediates specific El binding to the MO, thereby enhancing El-dependent viral DNA replication (Berg and Stenlund, 1997; Mohr et al, 1990). Interestingly, while oriP- containing plasmids are subject to once-per-cell cycle replication control that is coordinated with host chromosome replication, MME-containing plasmids replicate independently of host DNA during S phase using a poorly defined random-choice initiation mechanism (Gilbert and Cohen, 1987; Ravnan etal, 1995; Piirsoo et al, 1996). In addition to El, E2 and MO, long-term maintenance of BPV episomes in diving cells also requires the cis minichromosome maintenance element (MME), which is also found in the upstream regulatory region (URR) (Piirsoo et al, 1996). Indeed, in the presence of El and E2, MO and MME-containing plasmids are stable in mitotically active cells (Piirsoo et al, 1996). MME contains 12 binding sites for E2 (Li etal, 1989; Piirsoo et al, 1996) and this serves as the anchoring function for the E2 protein, which tethers MME-containing plasmids to mitotic chromosomes and allows for their stable persistence (Lehman and Botchan, 1998; lives et al, 1999). It has been shown that E2 and MME alone are sufficient for partitioning of plasmids that contain a heterologous replication origin in mouse cells (Silla et al, 2005). This situation is very much analogous to the ability of FR and EBNA1 to confer maintenance stability to plasmids that contain heterologous replication origins (Krysan and Calos, 1993; Wade-Martins et al, 1999; Kapoor et al., 2001; Silla et al, 2005). The viral E2 protein contains an N-terminal transactivation domain, which is also responsible for binding El (Berg and Stenlund, 1997; Chen and Stenlund, 1998; 2000; Gillitzer et al, 2000), an unstructured central hinge region, and a C-terminal domain, which is involved in DNA binding and dimerization and whose structure is similar to the DBDR of EBNA1 (Dostatni et al, 1988; Hegde et al, 1992; McBride et al, 1988; Bochkarev et al, 1995; see above). Two shorter versions of E2 also occur naturally in BPV1. They contain a C-terminal DNA binding
31 domain but lack the transcriptional activation domain; as such, they act as repressors of transcription and replication (Choe et at, 1989; Lambert et at, 1989; Chiang et at, 1992). Like EBV, BPV1 episomes are thought to partition faithfully to daughter cells by E2- mediated tethering to mitotic chromosomes. It has been shown by several independent investigators that BPV1 episomes and MME-containing plasmids stain mitotic chromosomes in a speckled pattern (Lehman and Botchan, 1998; Skiadopoulos and McBride, 1998; lives et at, 1999; Bastien and McBride, 2000). The transactivation domain (TA) of E2 is necessary and sufficient for such an association during mitosis (Skiadopoulos and McBride, 1998; Bastein and McBride, 2000). It has been noted that the BPV1 genomes also associate with interphase chromatin in a speckled fashion similar to the staining of mitotic chromosomes (lives et at, 1999). This observation led the authors to speculate that viral genomes may be placed on host DNA such that the viral episome is allowed access to the host replication machinery during S phase (lives et at, 1999). However, more investigation is required to support this hypothesis. Within the last few years, a large body of evidence has emerged implicating Bromodomain protein 4 (Brd4) as the mitotic tethering factor for E2. Using an affinity purification technique to identify protein partners of E2, You et al. (2004) discovered that Brd4 bound strongly to the viral protein and this interaction mapped to the C-terminal domain of Brd4 and the N-terminal domain of E2. The first clue that the E2-Brd4 association is crucial for BPV episome genome segregation came from the same group of investigators who showed that E2 and Brd4 co-localized on mitotic chromosomes in punctate spots (You et at, 2004). Moreover, expression of the C-terminal domain of Brd4 (Brd4-CTD) abrogated the co-localization of E2 and Brd4 on mitotic chromosomes, blocked the association of the viral episomes with Brd4, and resulted in the loss of BPV 1-based plasmids in cells (You et at, 2004). Other lines of evidence were later provided to support the role of Brd4 in E2-mediated attachment to mitotic chromosomes and cell transformation. First, E2 proteins that were defective in Brd4 binding were also unable to bind to mitotic chromosomes (Baxter et at, 2005). Second, ectopic expression of mouse Brd4 and E2 could reconstitute BPV 1-based plasmid maintenance in Saccharomyces cerevisiae using a plasmid loss assay similar to that used for the EBNAl/hEBP2 system described above (Brannon et at, 2005). Third, BPV1 E2 greatly stabilized the association of Brd4 with chromatin in mitosis, suggesting that E2 actively commandeers its chromosome-binding partner Brd4 (McPhillips et at, 2005). Fourth, disruption of the E2-Brd4
32 interaction by transfecting Brd4-CTD into BPV1-transformed cells significantly reduced the number of BPV1 genomes in cells compared to cells without Brd4-CTD (You et al, 2005). These results strongly supported the contention that the E2/Brd4 association is important in papillomavirus plasmid maintenance. Brd4 is a member of the BET family of proteins, which are characterized by the presence of two bromodomains (B) and an extra-terminal (ET) protein interaction domain. Found in many chromatin-associated proteins, transcription factors, and in nearly all known histone acetyltransferases, the bromodomain is an evolutionarily conserved module that binds acetylated histones. Brd4 preferentially binds acetylated lysine-14 (K14) on histone H3 and K5/12 on histone H4 (Dey et al, 2003). BET family members have been found to associate with chromatin during interphase, and for some family members, such as Brd2 and Brd4, the localization on DNA persists throughout mitosis (Dey et al, 2000; 2003). For this reason, Brd4 is also referred to as the mitotic chromosome-associated protein (MCAP) (Dey et al, 2000). Brd4 is considered as a "long" family member of the BET family of proteins because it contains an additional C-terminal "tail" and it is to this region of Brd4 that E2 binds (You et al, 2004). Brd4 has been implicated to have a role in RNA polymerase II-mediated transcription as mouse Brd4 interacts with the active form of positive transcription elongation factor b (p-TEFb) (Yang et al, 2005). The significance of the interaction of E2 with Brd4 is not limited to segregation, as considerable evidence points to a role Brd4 in E2-mediated gene expression. You et al (2004) found that a large amount of Brd4 complexed with E2 in asynchronous cells, while McPhillips et al. (2005) showed that the presence of E2 stabilized Brd4 association on chromatin not only during mitosis but also during interphase. These observations suggested that the E2-Brd4 complex is not only limited to episome partitioning during mitosis, but it could also be involved in other roles throughout the papillomavirus life cycle. Recent studies showed that Brd4 mediates the transcriptional activation function of E2 (lives et al, 2006; Schweiger et al, 2006; McPhillips et al, 2006). Expression of Brd4 CTD or down-regulation of Brd4 using RNAi impaired E2-dependent transcription of reporter plasmids and native promoters but E2-specific replication was unaffected (lives et al, 2006; Schweiger et al, 2006). Furthermore, alanine- scanning mutations within the N-terminal transcriptional activation domain of E2 which disrupted Brd4 binding also abrogated E2 transcription (Schweiger et al, 2006). McPhillips et
33 al (2006) showed that the E2-Brd4 interaction was not limited to BPV1 E2, but occurred with E2 viral proteins from a diverse group of both human and animal papillomaviruses. They discovered that expression of Brd4 CTD impaired transcription by all E2 proteins, implicating the importance of Brd4 in E2-mediated transcriptional activation. Conversely, it was shown that not all E2 proteins (particularly not the E2 proteins from the clinically relevant alpha papillomaviruses such as HPV-11 and HPV-31), co-localized with Brd4 on mitotic chromosomes (McPhillips et al, 2006). Moreover, for those E2 proteins that did not co-localize with Brd4, point mutations which disrupted Brd4 binding did not abolish E2 association with mitotic chromosomes, indicating that Brd4 is not involved in genome partitioning of all papillomaviruses (McPhillips et al, 2006). Indeed, the same authors noted that while all E2 proteins associated with chromosomes during mitosis, the staining pattern differed significantly depending on the papillomavirus type (Oliviera et al, 2006). Thus, E2 may utilize multiple chromosome-bound targets in order to ensure proper genome transmission and persistent infection. In support of this hypothesis, Parish et al (2006) recently discovered that the ATP-dependent DNA helicase, ChlRl and its yeast homologue Chllp, bound BPV1 E2 and played a crucial role in episome maintenance. E2 proteins that harboured a point mutation which disabled its ability to interact with ChlRl (but not with Brd4) did not associate with mitotic chromosomes and could not maintain the BPV1 viral genome even though replication was unaffected. Moreover, E2 and ChlRl were found to co-localize on mitotic chromosomes during prophase, after which E2 remained on chromosomes while ChlRl redistributed to the spindle poles. Silencing ChlRl resulted in the significant loss of E2 on mitotic DNA. From these observations, the authors proposed a more elaborate version of the mitotic tethering model to explain episome partitioning. They suggest that ChlRl is required for events that tether E2 onto mitotic chromosomes during the early stages mitosis. However, other host cellular proteins, such as Brd4, would be required to keep E2 properly tethered on sister chromatids until the end of mitosis.
1.3.2. Kaposi's Sarcoma-Associated Herpesvirus (KSHV)
Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) is a gammaherpesvirus of the rhadinovirus genus that is present in Kaposi's sarcoma (KS) and various lymphoproliferative diseases, such as primary effusion lymphoma (PEL), multicentric
34 Castleman's disease and AIDS-associated KS (reviewed in Schulz, 1998). Similar to EBV and PV infections, KSHV infection is predominantly latent where infected cells maintain approximately 40-80 copies of the double-stranded DNA genome stably in cells (Cesarman et al, 1995; Decker et al, 1996; Ballestas and Kaye, 2001). The c/s-acting terminal repeats (TR) located at the 5'end of the viral genome and the trans-acting factor encoded by open reading frame 73 (ORF73), referred to as latency-associated nuclear antigen-1 (also known as LANA1, LNA1, or LN1) (Kedes et al, 1997; Kellam et al, 1997; Rainbow et al, 1997), are the functional homologues of EBV oriP and EBNA1, respectively. Specifically, LANA1 is involved in latent DNA replication from the TR, transcriptional activation and repression, as well as KSHV episome persistence. These functions will be described briefly in this section with an emphasis on LANA1-mediated segregation. LANA1 has been shown to support the initiation of DNA replication of TR-containing plasmids (Grundhoff and Ganem, 2003; Hu et al, 2002; Lim et al, 2002). This is presumed to be accomplished by the interaction of LAN A1 with the origin recognition complexes (ORCs), both of which have been found on KSHV TR by ChIP experiments (Verma et al, 2006). Recent studies showed that replication of viral episomes requires the N-terminus (specifically amino acids 5-13) and the C-terminus of LANA1 (Barbera et al, 2004; Verma et al, 2006). The C- terminus is required for association of LANA1 with the TR sequences found within plasmids and KSHV episomes (Ballestas et al, 1999; Cotter et al; 2001). LANA1 is also a potent transcriptional regulator, as it has been shown to be capable of activating, as well as repressing, heterologous viral and cellular promoters (Renne et al, 2001; Garber et al, 2002; Viejo-Borbolla et al, 2003). Although the mechanism by which this occurs is unclear, it may involve the ability of LAN A1 to interact with cellular transcription factors such as CREB, CBP, Spl, p53, and pRB (An et al, 2002; Firborg et al 1999; Krithivas et al, 2000; Lim et al, 2000; Radkov et al, 2000). LANA1 represses transcription from the KSHV TR by binding to sequences within the TR via its C-terminal DNA-binding domain (Ballestas and Kaye, 2001; Cotter et al., 2001; Garber et al., 2002; Lim et al, 2002). For optimal transactivation of certain cellular promoters, however, the first 22 amino acids of LANA1 as well as its C-terminus are required (Wong et al, 2004). Thus, LANA1-mediated transcription is a complex process that occurs via multiple mechanisms and involves both the N-terminus and C-terminus of the viral protein where each domain is capable of performing various different functions.
35 The only viral components required for episome persistence are the KSHV TR and LANA1. It has been shown by multiple investigators that the presence of TR stabilizes plasmids in proliferating cells provided that LANA1 is also present (Ballestas et al, 1999; Ballestas and Kaye, 2001; Cotter and Robertson, 1999; Szekely et al, 1998), suggesting that LANA1 governs the replication and segregation of KSHV genomes. Moreover, deletion of LANA1 in the context of the KSHV genome (by transposon-mediated mutagenesis) or down-regulation of LANA1 (using hairpin silencing constructs) has been shown to lead to episome loss (Ye et al, 2004; Godfrey et al, 2005), further implicating LANA1 in stable episome maintenance. Depending on the viral isolate, the TR is comprised of 35-45 tandem copies of an 801 bp unit that contains an origin of replication and the cw-acting element required for episome maintenance (Lagunoff and Ganem, 1997, Ballestas et al, 1999; Ballestas and Kaye, 2001; Garber et al, 2001). However, only two such units placed in tandem are necessary and sufficient for episome persistence (Ballestas and Kaye, 2001; Lim et al, 2002). The mechanism by which KSHV mediates episome partitioning is thought to be through L AN A1-mediated association with mitotic chromosomes. LANA1 has been observed to stain mitotic chromosomes as discrete puncta that co-localize with the KSHV genome (Ballestas et al., 1999; Cotter and Robertson, 1999; Szekely et al, 1998). In addition, LANA1 may preferentially interact with heterochromatin DNA (Szekely et al, 1999; Mattsson et al, 2002). In the absence of viral DNA or TR-containing plasmids, the punctate staining pattern is not seen; instead, LANA1 diffusely coats mitotic chromosomes (Ballestas et al, 1999; Piolot et al, 2001; Viejo- Borbolla et al, 2003). This might indicate that the binding of LANA1 to its cognate binding sites changes the host chromosome association or simply that most of the LANA1 protein is sequestered on the TR sequences when KSHV plasmids are present so that little LANA1 is available to bind chromosomes individually. Moreover, this observation could also reflect the possibility that LANA1 has higher affinity for TR sequences than for chromosome. The C- terminus of LAN A1 contains the DNA-binding and dimerization domain responsible for sequence-specific contacts with the TR of the viral episome during both interphase and mitosis (Ballestas et al, 1999; Cotter et al, 2001; Garber et al, 2002; Lim et al, 2002; Komatsu et al., 2004). Interestingly, the DNA binding domains of LANA1 and EBNA1 are predicted to be structurally homologous since certain LANA1 residues important for DNA binding correspond
36 to those required for EBNA1 DNA binding (Grundhoff and Ganem, 2003; Kelley-Clarke et ah, 2007). In the context of full-length LANA1, it is the N-terminus of the viral protein, specifically amino acids 5 to 13, that mediates the predominant interactions with mitotic chromosomes since deleting these key residues abolished chromosome attachment and episome persistence (Piolot et ah, 2001; Barbera et ah, 2004). However, it has been shown that a (GFP)-tagged peptide of the LANA1 N-terminus (GFP-LANA1-N) and a GFP-tagged LANA1 C-terminus (GFP-LANA1-C) are each able to associate with mitotic DNA, suggesting that both termini contain chromosome- binding sites (Piolot et ah, 2001; Krithivas et ah, 2002). Interestingly, while GFP-LANA1-N stains chromosomes diffusely, GFP-LANA1-C formed punctate spots characteristic of full- length LANA1 (Schwam et ah, 2000; Krithivas et ah, 2002; Kelley-Clarke et ah, 2006). This suggests that the N-terminus of LAN A1 may associate with different protein partners compared to its C-terminus although in the context of the full-length protein, both sets of proteins may be utilized by LANA1 to form stable associations with DNA. LANA1 attachment to mitotic chromosomes has been proposed to be mediated by the following cellular proteins based primarily on their ability to interact with LANA1: histone HI, histones H2A and H2B, MeCP2, DEK, Brd2, and Brd4 (Cotter and Robertson, 1999; Barbera et ah, 2006; Krithivas et ah, 2002; Piatt et ah, 1999; You et ah, 2006). In brief, Brd2, a BET family protein like Brd4, is a serine/threonine kinase that interacts preferentially with acetylated K12 on histone H4 and it has roles in cell-cycle regulation and transcription (Denis et ah, 2006 and references therein). MeCP2 binds methylated CpG islands and it has been proposed to have roles in chromatin compaction and transcriptional repression (reviewed in Adams et ah, 2007). DEK is a DNA-binding phosphoprotein involved in chromatin organization, mRNA processing, and transcription (Faulkner et ah, 2001; Hollenbach et ah, 2002; Waldmann et ah, 2003). It should be noted at this time that while studies (discussed below) implicate these proteins in episome persistence, the lack of any functional tests that directly assess their importance in this role means that LANAl's mitotic tethering partner(s) is still unclear. In KSHV-positive body cavity lymphoma cell lines, Cotter and Robertson (1999) discovered that LANA1 was able to efficiently co-precipitate with histone HI. Furthermore, mitotic chromosome association of LANA1 can be rescued by replacing the N-terminus of LANA1 with histone HI (Shinohara et ah, 2002). These results implicate histone HI as a target
37 for LANA1-mediated tethering of KSHV genomes onto host chromosomes. However, affinity chromatography experiments using GFP fused to LANA1 residues 1 to 32, which mediate the predominant interactions with mitotic chromosomes, could not detect this interaction (Barbera et al, 2006). Thus, the function of the LANA1-H1 interaction remains elusive. Barbera and colleagues (Barbera et al, 2006) identified histones H2A and H2B as strong interacting partners of LANA1. Subsequent studies showed that full-length LANA1 substituted at residues essential for chromosome binding could not bind the core histones, suggesting that LANA1 interactions with histones H2A and H2B were required for chromosome association. LANA1 was found to bind specifically and directly to the folded domain as opposed to the histone tails of H2A and H2B since tailless H2A-H2B histones were still capable, of interacting with LANA1. Indeed, an X-ray crystal structure of LANA1 residues 1-23 in complex with the core nucleosome showed that the LANA1 hairpin fit the acidic pocket formed by the folded domains of histones H2A-H2B. Thus, the authors proposed that histones H2A-H2B form a docking site for LANA1, which is essential for episome persistence. However, it is not clear that the H2A-H2B interface would be accessible to LANA1 during mitosis, when the chromosome is highly compacted. In addition, since H2A-H2B is highly conserved among higher eukaryotes, this chromosome attachment mode would not explain why the interaction of LANA1 with chromosomes is species-specific (i.e. LANA1 binds human chromosomes but not murine chromosomes (Krithivas etal, 2002)). In fact, given the roles of LANA1 in regulating gene expression and replication, it is tantalizing to suggest that the interactions with histones could reflect transcriptional or replication functions and not necessarily segregation. Nevertheless, it is also possible that one interacting protein can mediate multiple LANA1 functions. Co-IP experiments revealed that methyl CpG binding protein (MeCP2) was able to interact with wild-type LANA1 (wtLANAl) but not with a mutant of LAN A1 that lacks the first 15 amino acids (LANA1AN) (Krithivas et al, 2002). The expression of FLAG-tagged human MeCP2 (FLAG-hMeCP2) in a mouse cell line that expressed low levels of MeCP2 resulted in the localization of GFP-tagged LANA1 (GFP-LANA1) to heterochromatin regions of DNA during interphase and mitosis. In contrast, in the absence of FLAG-hMeCP2 or when GFP- LANA1 AN was co-transfected with hMeCP2, LANA1 association with heterochromatin was not observed. These results suggested that MeCP2 may govern LANA1 targeting to heterochromatin regions of DNA. The same group also identified DEK as a binding partner of
38 the C-terminal domain of LANA1 (LANA1-C) (Krithivas et al, 2002). They showed that in the presence of myc-tagged DEK (Myc-DEK), but not in its absence, GFP-LANA1AN localized to mouse and human chromosomes, suggesting that DEK is able to target LANA1 through its C- terminal domain to cellular DNA. While MeCP2 and DEK may function to tether LANA1 to cellular chromosomes, the importance of this function with respect to stable episome persistence in proliferating cells remains to be tested. The C-terminus of LAN A1 has been shown to bind the conserved ET domain of Brd2 (Piatt et al, 1999; Viejo-Borbolla et al, 2005; You et al, 2006). Subsequent immunofluroescence studies using Brd2-specific antibodies revealed little co-localization between LANA1 and Brd2 on mitotic chromosomes of KSHV-infected cells (You et al, 2006). However, it was noted that in KSHV-negative cells, Brd2 staining of mitotic chromosomes was significantly reduced compared to KSHV-positive cells, suggesting that LANA1 may increase Brd2 recruitment to host chromosomes (Mattsson et al, 2002; You et al, 2006). It has also been suggested that the interaction between LANA1 and Brd2 may be important in KSHV latent origin of replication since Brd2 has been found to interact with TR through ChIP experiments (Stedman <*«/., 2004). LANA1 has also been shown to interact with Brd4 both in vivo and in vitro and the association occurs between the C-terminus of LANA1 and the ET domain of Brd4 (You et al, 2006; Ottinger et al, 2006). Moreover, in cells carrying an artificial KSHV episome or in KSHV-infected cells, Brd4 and LANA1 were observed to co-localize on mitotic chromosomes with a strong punctate staining pattern (You et al, 2006). This observation raised the possibility that interactions with Brd4 by viral proteins such as LANA1 and E2 may be a conserved mechanism used by viruses to ensure stable genome persistence. However, since the N-terminus of LANA1 is considered to be the dominant chromosome-binding region in the context of the full-length protein while the C-terminus of LANA1 to which Brd4 binds constitutes an independent chromosome-binding site, the function of the LANA1-Brd4 interaction in KSHV- infected cells still remains unclear. Nevertheless, it is still possible that in the context of full- length LANA1, the C-terminus can contribute to stable chromosome attachment. It is also worth mentioning that such an interaction may also be involved in LANA1-mediated transcriptional activities as seen for BPV1 (see Section 1.3.1.).
39 1.3.3. Herpesvirus Saimiri (HVS)
Herpesvirus saimiri (HVS) is a close primate relative of KSHV, as it is also a gammaherpesvirus of the Rhadinovirus genus. HVS naturally infects the squirrel monkey {Saimiri sciureus) without causing any obvious symptoms of disease (Melendez et ah, 1968). However, experimental infection of other primates can lead to a range of lymphoproliferative disorders within a few weeks (Fleckenstein and Desrosiers, 1982). The genomes of HVS and KSHV are highly similar in that of the 81 ORFs encoded by KSHV, at least 66 share significant homology with the ORFs found within HVS (Russo et ah, 1996). During latency, only three genes are expressed (Hall et ah, 2000a); they are ORF71, ORF72, and ORF73, all of which are also expressed in KSHV latency (Dittmer et ah, 1998; Talbot et ah, 1999). ORF71 encodes a cellular homologue of an anti-apoptotic FLICE inhibitory protein (Thome et ah, 1997), while ORF72 encodes a cyclin D homologue (Chang et ah, 1996). HVS ORF73 is a 64 kDa nuclear protein (Hall et ah, 2000b) which shares limited sequence homology with LANA1 but the two proteins possess significant structural homology. Both proteins contain a small N-terminal domain, a larger C-terminus, and a central, repetitive acidic region whose length varies between viral isolates (Hall et ah, 2000b; Verma and Robertson, 2003). The amino terminus of ORF73 contains two nuclear localization signals and the C-terminus is required for mitotic chromosome association, which occurs in a speckled pattern even in the absence of HVS episomes (Hall et al, 2000b; Verma and Robertson, 2003; Calderwood et al., 2004). A closer examination of the C-terminus of ORF73 (ORF73C) revealed that it contains two chromosome-association sites, CAS 1 and CAS2 (Calderwood et al., 2004). CAS1 contains a proline-lysine-lysine (PKK) motif which, if mutated, results in loss of chromosome association. The PKK motif can be found in the DNA binding domains of histone HI and H2 as well as in hEBP2. CAS2 is responsible for the multimerization of ORF73. Deletion of CAS2 disables multimerization and chromosome attachment, suggesting that multimerization is a prerequisite to DNA binding (Calderwood et ah, 2004). In addition to being a structural homologue of LANA1, HVS ORF73 is also a functional homologue. ORF73 alone is sufficient for the maintenance of plasmids containing HVS terminal repeats (Collins et ah, 2002; Collins and Medveczky, 2002; Verma and Robertson, 2003). It has been proposed based on several lines of evidence that the mechanism of episome maintenance is also mitotic tethering. First, ORF73 has been shown to bind specific sequences found within the
40 terminal repeat element of HVS (Verma and Robertson, 2003). Second, ORF73 is able to interact with mitotic chromosomes (Verma and Robertson, 2003; Calderwood et al, 2004). Third, metaphase spreads of HVS-transformed marmoset T cells have shown that HVS- based cosmid DNA co-localizes with ORF73 (Verma and Robertson, 2003). The cellular protein to which ORF73 binds on mitotic chromosomes is unknown although several potential candidates are currently being investigated by Whitehouse et al, including several members of the bromodomain family. The similarities between KSHV LANA1 and HVS ORF73 viral proteins prompted Griffiths and Whitehouse (2007) to investigate the ability of MeCP2 and DEK to mediate HVS episome maintenance. The authors discovered that MeCP2 but not DEK could interact with ORF73. Moreover, silencing MeCP2 resulted in the loss of HVS-based plasmid maintenance in mammalian cells compared to non-silenced cells. However, as the authors did not address whether silencing MeCP2 affected episome replication nor did they address if ORF73 dissociated from mitotic chromosomes upon MeCP2 silencing, it is unclear if MeCP2 is directly involved in ORF73 attachment to mitotic DNA and episome persistence. The authors did note, however, that MeCP2 is able to interact with ORF73 deletion mutants that cannot bind mitotic chromosomes. Moreover, they mention that there is no significant difference between ORF73 binding to murine mitotic chromosomes compared to human mitotic chromosomes even though the former cell line expressed reduced MeCP2 levels. Thus, based on these additional observations, the authors proposed that MeCP2 is likely involved in enhancing or stabilizing ORF73 binding to DNA as opposed to directly mediating DNA associations. Therefore, the mechanism by which HVS mediates episomal tethering still remains a mystery although the search for the cellular protein involved in mediating ORF73 attachment to mitotic chromosomes is actively being pursued.
1.4. Thesis Rationale
Considerable evidence indicates that low-copy number circular viral DNA molecules are able to persist indefinitely in proliferating cells due to a partitioning mechanism which likely involves tethering to cellular chromosomes in mitosis. In each case this involves an interaction of the viral origin binding protein with cellular proteins on the chromosomes. For BPV1, E2 appears to associate with mitotic chromosomes by interacting with Brd4 and ChlRl (see section 1.3.1). KSHV LANA1 has several candidate mediators for cellular DNA attachment, including
41 Brd2, Brd4, DEK and MeCP2 (see section 1.3.2), while HVS ORF73 has been predicted to associate with host chromosomes by interacting with MeCP2 (see section 1.3.3). Studies in our laboratory have shown that the tethering of EBV plasmids to mitotic chromosomes involves an interaction between EBNA1 and the cellular hEBP2 on mitotic chromosomes. However, it is not known if additional cellular proteins might also be important for EBNA1 to interact with chromosomes, either in conjunction with hEBP2 or in a parallel pathway. Indeed, the studies on BPV1 and KSHV suggest that more than one cellular protein is involved in tethering the viral genomes to the chromosomes. The fact that the Brd proteins and MeCP2 are implicated in the segregation of more than one virus suggests that there may be common cellular proteins that are utilized by multiple viruses to assure persistent infection, despite the lack of sequence homology between the viral origin binding proteins. The purpose of my research was to investigate the possibility that Brd2, Brd4, DEK and MeCP2 might have roles in EBNA1-mediated segregation of EBV-based plasmids. I tested this possibility using the yeast-based EBV plasmid loss assay previously developed in our laboratory that initially identified the role of hEBP2 in EBNA1-mediated segregation. I found that Brd4 enabled EBNA1 to segregate EBV-based plasmids in yeast, while Brd2 and MeCP2 had a general stimulatory effect on the segregation of all plasmids tested with or without EBNA1. I further identified the region of EBNA1 that was needed to function with Brd4 in the yeast system and showed that this region also mediates a Brd4 interaction in human cells. Finally, I examined the effect of silencing Brd2, Brd4 and MeCP2 on EBNA1 association with chromosomes in human cells.
42 II. MATERIALS AND METHODS
11.1. Plasmid Loss Assay Constructs
The maintenance stability of three different segregation test plasmids was monitored in the plasmid loss assay. These plasmids included pRS314, YRp7 and YRp7FR. pRS314 is a yeast centromeric plasmid that contains the ARSH4 and CEN6 elements and the TRP1 selection marker (Sikorski and Hieter, 1989). Since pRS314 can undergo autonomous replication and segregation, it was used in the plasmid loss assay as the "True Positive" control. YRp7 is a plasmid that contains the ARS1 element and the TRP1 selection marker (Stinchcomb et al, 1979). Since YRp7 can only undergo autonomous replication but not segregation, it is readily lost in yeast cells and was therefore used in the plasmid loss assay as the "True Negative" control. The construction of YRp7FR is described in Kapoor et al. (2001). In brief, the FR segregation element of EBV was inserted into YRp7 so that its proper partitioning would depend on EBNA1. EBNA1 and the EBNA1 mutants EBNA1A325-376, EBNA1A8-67, and EBNA1A61-83 were inserted into the low-copy number yeast expression plasmid p416MET25. Expression of EBNA1 is under control of the MET promoter. p416MET25 contains the CEN6/ARSH4 element and the URA3 selection marker (Mumberg et al, 1994). The construction of p416MET25.EBNAl and p416MET25.EBNAl A325-376 is described in Kapoor et al (2001) while construction of p416MET25.EBNAl A8-67 and p416MET25.EBNAlA61-83 is described in Wu et al. (2002). hEBP2, DEK, Brd2 and MeCP2 were inserted downstream of the PGK promoter of the pR425/PGK plasmid. pR425/PGK is a high-copy number, 2-micron expression plasmid that contains the LEU2 marker (Marcus et al, 1995). The construction of pR425/PGK.hEBP2 is described in Kapoor et al (2001). The cDNA clones for Brd2 (MGC:74927 IMAGE:6181728; provided as pCMV-SPORT6.Brd2), DEK (MGC:29866 IMAGE:5122743; provided as pCMV- SPORT6.DEK) and MeCP2 (MGC: 10245 IMAGE:3956518; provided as pOTB7.MeCP2) were purchased from the Mammalian Gene Collection (MGC) IMAGE consortium distribution network. The genes were amplified by polymerase chain reaction (PCR) with VENT DNA polymerase (New England BioLabs #M0254) using the following primers:
43 Brd2 N: 5' GGA AGA TCT GGA TCC ATA TGC TGC AAA ACG TGA CTC C 3' Brd2 C: 5' GGA AGA TCT GAA TTC TTA GCC TGA GTC TGA ATC AC 3'
DEK N: 5' GGA AGA TCT GGA TCC ATA TGT CCG CCT CGG CCC 3' DEK C: 5' GGA AGA TCT GAA TTC TCA AGA AAT TAG CTC TTT TAC AG 3'
MeCP2 N: 5' GGA AGA TCT GGA TCC ATA TGG TAG CTG GGA TGT TAG G 3' MeCP2 C: 5' GGA AGA TCT GAA TTC TCA GCT AAC TCT CTC GGT C 3'
Each PCR reaction was initiated with a "hot start" procedure where the mixture was heated to 94°C for 3 minutes. Following this, the reactions went through 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C (for DEK) or 53°C (for Brd2 and MeCP2) for 30 seconds, and primer extension at 72°C for 1.5 minutes (for DEK) or 2.5 minutes (for Brd2 and MeCP2).' At the end of the 30 cycles, the reaction was allowed an additional extension for 10 minutes at 72°C before finally coming to rest at 4°C. All PCR reactions were run on a 1% agarose gel and the appropriate PCR product was excised and gel-purified using Qiagen's Gel Extraction Kit. Purified PCR reactions were digested with BgllldX 37°C overnight and cleaned up using Qiagen's PCR Clean-Up Kit as per the manufacturer's protocol. The empty vector p425/PGK was also digested with BgUITor 2 hours at 37°C and cleaned up using Qiagen's PCR Clean-Up Kit. The ligations were conducted at a ratio of 3:1 (insert:vector), incubated overnight at 15°C, used to transform bacterial strain DH5a and plated on ampicillin (Amp)-containing LB plates (34 l^g/mL of Amp). The correct clones for p425PGK.DEK, p425PGK.Brd2, and P425PGK.MeCP2 were determined by restriction enzyme analysis and sequencing. Mouse Brd4 (mBrd4) was inserted into the pADNS yeast vector and its construction was previously described in Brannon et al, 2005. pADNS is a high-copy number, 2-micron expression plasmid that contains the LEU2 marker (Colicelli et al., 1989).
II.2. Yeast Plasmid Loss Assay
The yeast plasmid loss assay was performed as previously described (see Kapoor et al., 2001); however, a few modifications were made. Yeast strain YPH499 (described in Table 1) was transformed to uracil prototrophy with p416MET25.EBNAl, p416MET25.EBNAl A325-
44 Table 1. Yeast strains used in this study.
Strain Genotype Reference
MATa ura3-52 lys2-801 ade2-101 YPH499 Sikorski and Hieter, 1989. trplA63 his3A200 leu2Al
45 376, p416MET25.EBNAlA8-67, p416MET25.EBNAlA61-83 or empty vector p416MET25; to leucine prototrophy with P425/PGK.hEBP2, p425/PGK.DEK, p425/PGK.Brd2, p425/PGK.MeCP2, empty vector p425/PGK, pADNS.mBrd4 or empty vector pADNS; and to tryptophan prototrophy with the segregation test plasmids YRp7FR, YRp7 or pRS314. High- efficiency transformations were performed using a modification of the lithium acetate (Li Ac) method developed by Gietz and Schiestl (1995). For one transformation, YPH499 was grown in 5 mL of YPD complete medium (1% yeast extract/1% peptone/2% dextrose and 2% agar where neeeded) at 30°C for 3-4 hours until the culture reached an OD600 (optical density) between 0.7- 1.0. Cells were pelleted by low-speed centrifugation (1500 x g), washed with doubled distilled autoclaved water (ddFbO), resuspended in 1 mL of 0.1M LiAc and incubated in a 30°C water bath for 15 minutes. Cells were pelleted and the following components were added in following order: 240 |aL of 50% PEG 3500, 36 |^L of 1.0M LiAc, 50 uL of 2 mg/mL denatured salmon sperm DNA, and 34 uL of ddH^O containing a maximum of 2 pg of total DNA plasmid. The mixture was vortexed vigorously until the pellet was well resuspended, incubated at 30°C for 30 minutes, followed by heat shock at 42°C for 1 hour. After heat shock, cells were pelleted, resuspended in 100 uL of ddHbO and plated on synthetic complete (SC) plates (Sherman et al, 1979) that lack the appropriate amino acid in order to select for positive transformants containing the plasmid(s) of interest. For example, when all three of the aforementioned plasmids were transformed into YPH499, the transformation was plated on SC media lacking uracil, leucine and tryptophan (SC-Ura,Leu,Trp). Synthetic complete media contains 1.7 g/L of yeast nitrogen base without added amino acids and ammonium sulphate (Difco, 233520), 5 g/L of ammonium sulphate, and 2% agar where needed. The final amino acid concentrations were as follows: 20 mg/L L-histidine, 40 mg/L L-adenine, 40 mg/L L-tryptophan, 30 mg/L L-lysine, 30 mg/L L- leucine, and 20 mg/L L-uracil. Approximately 15-20 positive transformants of approximately equal size from the selective plates and with respect to the segregation test plasmid (SC-Ura,Leu,Trp) were inoculated into 5 mL of selective media and grown overnight (-16-20 hours) at 30°C while shaking in an aerated shaker at 200 rpm. The following day, cells growing in mid-log phase (OD600 between 0.5-1.0) were diluted into 5 mL of non-selective media (SC-Ura,Leu) such that OD600 equals -0.001. Yeast cultures were grown at 30°C for -25 generations (-60 hours). During this out-growth period, it was necessary to repeat the dilutions twice more to
46 ensure that cells were maintained in log-phase. At the end of the non-selection period, 10 uL of each culture was spotted in 10-fold serial dilutions on selective and non-selective plates such that the least dilute culture had an OD6oo of 0.1. In parallel, approximately 200-300 colonies were spread on selective and non-selective plates. Plates were transferred to 30°C incubator and allowed to grow for 3 days. Colonies that grew on selective and non-selective plates were counted and a ratio of colony growth on selective versus non-selective media was taken as a measure of how well the segregation test plasmid was maintained in yeast cells. Standard two- tailed T-tests were performed using the "True Positive" control as the sample to which all other test conditions are compared (unless stated otherwise). The significance threshold was set at p<0.01.
II.3. Co-immunoprecipitation Constructs
pGEX-6P-l hBrd4 full-length clone (previously described in You et al, 2004) was purchased from Addgene (plasmid 14447, visit www.addgene.org/14447). pcDNA3.hBrd4 was kindly made by our lab technician Tin Nguyen. In brief, pGEX-6P-l hBrd4 and empty vector pcDNA3 were digested with BamHI and NotI and the digestion products were run on a 1% low- melt agarose gel. The appropriate hBrd4 fragment (~4.5 kb) and linearized pcDNA3 vector (~5.4 kb) were excised from the gel, melted at 50°C, and combined in a ligation reaction at a molar ratio of 3:1 (insert:vector). Ampicillin-resistant colonies were screened by restriction enzyme analyses for the correct pcDNA3.hBrd4 construct and later sequenced. Brd4 is expressed from the high-expression cytomegalovirus (CMV) promoter. The construction of the EBNA1-expression plasmids used in the co-immunoprecipitation experiments is described in Wu et al. (2002). These constructs include: pc3oriPEBNAl Agly-ala (wild-type EBNA1), pc3oriPA325-376 (EBNA1 lacking amino acids 325-376), pc3oriPA8-67 (EBNA1 lacking amino acids 8-67), pc3oriPA8-67/325-376 (double deletion mutant of EBNA1 lacking amino acids 8-67 and 325-376), pc3oriPA61-83 (EBNA1 lacking amino acids 61-83), and pc3oriP452-641 (which contains the C-terminal DNA binding domain of EBNA1 spanning amino acids 452-607). All EBNA1 constructs are expressed from the high-efficiency CMV promoter.
47 II.4. Co-immunoprecipitation in 293T Cells
Approximately 7-8 x 105 293T cells were seeded in 10-cm dishes such that after overnight growth (-20-24 hours) at 37°C in DMEM complete media (DMEM from SIGMA supplemented with 10% fetal bovine serum albumin (FBS), 1% L-glutamine and 1% penicillin/streptomycin), cells were -50-60% confluent and ready for transfection. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent as per the manufacturer's protocol. In general, 2 uL of Lipofectamine 2000 was used per (ig of DNA being transfected. Since each EBNA1 mutant was observed to express at different levels in human cells, the amount of DNA being transfected per 10-cm dish varied depending on the EBNA1 mutant. For the high-expressing pc3oriPEBNAl, pc3oriPA325-376 and pc3oriPA61-83 EBNA1 constructs, 0.5 ug of DNA was used per 10 cm dish; for the low-expressing pc3oriPA8-67 EBNA1 construct, 6.0 ug of DNA was used; and for the moderate-expressing pc3oriPA8-67/325-376 and pc3oriP452-641 constructs, 4.0 ug of DNA was used. Where hBrd4 was over-expressed, 2 ug of pcDNA3.hBrd4 per 10-cm dish was co-transfected with the EBNA1-expression plasmids. Approximately 24 hours after transfection, cells were transferred from 10-cm dishes to 15-cm dishes and allowed to continue growth in DMEM complete media for 3 days at 37°C. Transfected 293T cells were harvested, washed in cold phosphate buffered saline (PBS) and lysed in at least 5-times the pellet volume with modified RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 0.5% sodium deoxycholate, protease inhibitor cocktail (ROCHE), and ImM PMSF). Cells were lysed on ice for 30 minutes and then digested with 10 units/mL of DNasel (Fermentas, Cat #EN0521) for 30 minutes at room temperature. Lysates were spun down at high speed (>16,000g) for 20 minutes at 4°C and the clarified lysate was pre-cleared with 50 uL/mL of protein A-agarose beads (Santa Cruz, Cat #sc-2001) for at least 2 hours while rotating at 4°C. The beads were collected by centrifugation for 2 minutes at 2,400 rpm and the concentration of the pre-cleared lysate was determined by Bradford reagent (Bio-Rad, Canada). For each clarified and pre-cleared whole-cell lysate, 1 mg of total protein was used in the immunoprecipitation reaction. The volume of the reaction was adjusted so that all lysates had the equivalent protein concentration of 3.0 mg/mL. These lysates were incubated overnight with the protein-A agarose beads with or without the coupled Brd4 antibody (see blow) while rotating at 4°C.
48 Protein A-agarose beads were coupled to Brd4 antibodies in the following manner before they were added to the pre-cleared lysates: 30 uL of protein A-agarose beads was incubated in 500 uL of cold PBS in the presence or absence of 1.0 uL of rabbit polyclonal anti-Brd4 antibody (Abeam Cat#46199) for at least 4 hours while rotating at 4°C. The beads were then blocked with lmg/mL of bovine serum albumin (SIGMA) for at least 1 hour while rotating at 4°C. The beads were washed once with cold PBS and then added to the appropriate lysate for incubation. After overnight incubation of the lysate with beads, the beads were harvested by centrifugation for 2 minutes at 2,400 rpm and the supernatant was carefully aspirated, leaving the beads at the bottom of the eppendorf tube. Beads were washed thrice with 1 mL of cold modified Buffer A while inverting the eppendorf tubes 20 times during each wash. The protein complexes were eluted from the beads with 40 uL of 2X SDS loading buffer and boiled for 10 minutes prior to loading on 8% SDS-PAG (polyacrylamide gel). For input, 50 ug of each lysate (equivalent to 5% of total whole-cell protein used in the immunoprecipitation reactions) was also loaded on an 8% SDS-PAG. EBNA1 and Brd4 proteins were detected by Western blot analyses using specific antibodies (see Sections II.8 and II.9).
II.5. Silencing Constructs for hBrd4 (esiBRD4), hBrd2 (siBRD2) and hMeCP2 (siMecP2)
Endoribunuclease-prepared short interfering ribonucleic acids (esiRNAs) were prepared for the long-isoform of hBrd4 (henceforth referred to as esiBRD4) in Dr. Lawrence Pelletier's laboratory at Mount Sinai Hospital. The sequence for hBrd4 (NM058243) was uploaded into the web-based DEQOR program (freely available at http://cluster-l.mpi- cbg.de/Deqor/deqor.html), which selects a region within the gene that has optimal silencing potential and that has less than 5% off-target silencing effects (see Henschel et al, 2004 for review). For hBrd4, a 392 bp region was chosen by DEQOR which targets the 5' region of the gene, spanning nucleotides 153-508. The protocol for the generation of esiRNAs has been previously described (Yang et al, 2002). In brief, the 392 bp region of hBrd4 was amplified by PCR using the following forward (Fwd) and reverse (Rev) primers, each of which contains the T7 promoter sequence (underlined) at the 5' end:
esiBRD4 Fwd: 5' TCACTATAGGGAGAGCAACCCTAACAAGCCCAAGA 3' esiBRD4 Rev: 5' TCACTATAGGGAGACCGGTTTCTTCTGTGGGTAGC 3' The PCR product from the first round (PCR#1) was subsequently used in a second round of PCR reaction (PCR#2) to further amplify the segment of interest. The following standard T7 primers were used:
T7 prom Fwd: 5' GCTAATACGACTCACTATAGGGAGAG 3' T7 prom Rev: 5'GCTAATACGACTCACTATAGGGAGAC 3'
A small fraction of PCR#2 (9%) was in vitro transcribed with T7 polymerase overnight at 37°C for 12-16 hours. The single-stranded RNA (ssRNA) transcripts were annealed into double- stranded RNA form (dsRNA) and then digested into 18-25 bp oligomers with RNAselll. The newly generated esiBRD4 was isolated from the reaction mixture using a Q-Sepharose slurry and then purified by standard isopropanol precipitation protocol. Generated esiBRD4 was checked on a 4% agaorse gel to ensure its purity. The working stock of esiBRD4 was 100 ng/ul and was diluted using RNase-free water. Brd2 small-interfering ribonucleic acids (siBRD2) was selected and purchased from Invitrogen's Stealth Select RNAi collection (primer name BRD2-HSS109273). Its oligonucleotides comprised the sequence 5'-UUCAACUCUCCUCUUAAUAGUACCC-3' and the respective complimentary siRNA sequence 5'-GGGUACUAUUAAGAGGAGACUUGAA- 3'. MeCP2 siRNA (siMeCP2) was custom-made and purchased from Invitrogen based on sequences previously published by Mann et al. (2007). The oligonucleotides comprised the sequence 5'-CGUGAAGGAGUCUUCUAUCCGAUCU-3' and the respective complimentary siRNA sequence 5'-AGAUCGGAUAGAAGACUCCUUCACG-3'. For control, siRNA targeting GFP (siGFP) was custom-made and purchased from Invitrogen and it comprised the sequence 5'-GCAAGCUGACCCUGAAGUUCAU-'3' and the respective complimentary siRNA sequence 5'-GAACUUCAGGGUCAGCUUGCCG-3'. All siRNAs were used at a working stock concentration of 20 uM, which is equivalent to -150 ng/ul.
II.6. Silencing hBrd4, hBrd2 and hMeCP2 in HeLa.EBNAl Cells
HeLa cells expressing EBNA1 were generated as follows: Approximately 8xl05 HeLa cells were seeded on 10-cm dishes in DMEM media supplemented with 10% FBS and 1% L-
50 glutamine. The following day, HeLa cells that were -50-60% confluent were transfected with 6 ug of pc3oriPEBNAl using 12 ul of FuGENE 6 Transfection Reagent (Roche). 24 hours later, the media was changed to complete DMEM media, and on the following day, cells were transferred to 15-cm dishes at which time stable transformants containing pc3oriPEBNAl were selected using 300 ul of Geneticin/G-418 (Invitrogen, Cat no. 10131-027, supplied at 50 mg/mL stock solution) drug per 20 ml of media. From this point onward, HeLa cells containing EBNA1 (denoted as HeLa.EBNAl) were constantly kept under selection for up to 4 weeks. Silencing experiments were done on cells that had been under selection for 2-3 weeks. However, during the silencing procedure, drug selection was removed. The silencing experiments were performed as follows: On day 1, ~6.7-7xl05 HeLa.EBNAl cells were seeded on 10-cm dishes. On day 2, 12 ul of esiBRD4, 12 ul of siBRD2, 12 ul of siMeCP2 or 6 ul of siGFP were used to transfect HeLa.EBNAl cells at -40-50% confluency. Each microlitre of the silencing construct used was delivered into cells with 2 ul of Lipofectamine 2000. On day 3, the silencing procedure was repeated at which point the HeLa.EBNAl cells were -60-70% confluent. On the fourth day, one quarter of the cells was plated in 6-well format to check for silencing efficiency and the rest was transferred to 15-cm dishes. If mitotic cells were required for the following day, then colcemid (Invitrogen, Cat. No. 15212012) was added at a final concentration of 0.1 ug/ml medium and cells were incubated overnight for -14-16 hours. Prior to the addition of colcemid to the cells, it was important to ensure that cells had spread evenly at the bottom of the culture dish. This took -6 hours after transferring cells to new plates.
II.7. Biochemical Fractionation
Mitotic cells were harvested by mitotic shake-off and the remaining G2-phase cells were trypsinized and collected. Log-phase cells were also harvested by trypsinization. All cells were washed twice in 1 mL of cold PBS. Equal cell numbers from each sample (~1.5xl06 cells) were lysed in 100 ul of Buffer A (20 mM Tris HC1 [pH 7.5], 15 mM KC1, 30 mM MgCl2, 0.1% NP-40, 1 mM dithiothreitol, 0.5 mM EDTA,1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine and IX protease inhibitor cocktail [Roche Diagnostics]) on ice for 30 minutes. Fifty microlitres of the cell lysate was removed (sample W), and the remaining 50 ul of lysate was spun at 10,000 rpm for 10 min at 4°C in a microcentrifuge. The supernatant (sample S) was removed and
51 transferred to fresh eppendorf tubes at which point 2 ul of Buffer A was added to account for the decrease in volume due to the loss of the pellet. The pellet was resuspended in 48 ul of Buffer A (sample P) instead of 50 ul since the pellet size corresponded to ~ 2 ul. To both samples W and P, 1 ul or 1 unit of DNasel (Fermentas, Cat #EN0521) was added and the samples were incubated at room temperature for 30 minutes. Finally, SDS sample buffer was added to all three fractions and boiled for 10 minutes. After a quick spin, equal volumes from each sample representing equal cell numbers were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose membrane. The membranes were probed for the proteins of interest as described below.
11.8. Antibodies
The primary antibodies used in these studies and their corresponding dilutions were as follows: mouse monoclonal anti-EBNAl (OTlx) used at 1:5000; rabbit polyclonal anti-hEBP2 used at 1:5000 (as described in Wu et al, 2000); home-made rabbit polyclonal anti-Nap 1 serum used at 1:10000; goat polyclonal anti-Mcm2 used at 1:5000 (Santa Cruz sc-9839); rabbit polyclonal anti-Brd4 used at 1:5000 (Abeam ab46199); goat polyclonal anti-Brd used at 1:1000 (Abeam ab3718); rabbit polyclonal anti-MeCP2 used at 1:1000 (Abeam ab2828); rabbit polyclonal anti-DEK used at 1:1000 (Santa Cruz sc-30213); mouse monoclonal anti-yeast actin used at 1:500 (Abeam ab8224); and mouse monoclonal anti-actin used at 1:20000 (Calbiochem, Cat. No. CP01). Secondary polyclonal goat anti-mouse (sc-2005), goat anti-rabbit (sc-2004), or donkey anti-goat antibodies (sc-2020) conjugated to horseradish peroxidase (HRP) were all purchased from Santa Cruz Biotechnology Inc. and used at 1:5000. All antibodies were diluted in 5% milk/PBS buffer.
11.9. SDS-PAGE and Western Blot Analysis
To check silencing efficiency, cells were harvested by trypsinization using Trypsin- EDTA solution as per the manufacturer's protocol (Sigma-Aldrich, Cat. No. T3924). Cells were washed with cold PBS, and lysed for 30 minutes at 4°C in lysis buffer (25 mM Tris pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5% NP-40,1 mM DTT, and complete EDTA-free protease inhibitor cocktail [Roche Diagnostics, Cat. No. 11873580001]). The whole-cell extract was spun for 20
52 minutes at 13,200 rpm (>15,000g) in a microcentrifuge and the supernatant was transferred to a new eppendorf tube. The protein concentration was determined using Bradford reagent (Bio- Rad, Canada). Aliquots containing equal amounts of total protein (usually 50 ug) were boiled in SDS sample buffer and resolved by 8% SDS-PAG electrophoresis (SDS-PAGE). Gels were run at 100V for-1.5 h before they were transferred in transfer buffer [20% methanol, 25 mM Tris (Bioshop TRS001) and 0.19 M glycine (Bioshop GLN001)] onto nitrocellulose membrane (Whatman, Cat. No.W-10439196) for 3 hours at 60V or overnight at 35V. Membranes were first blocked for 1 hour at room temperature in 5% milk/PBS, and then incubated in the appropriate primary antibody diluted in 5% milk/PBS for 1 hour at room temperature. After three 15 minutes washes in PBS, the membranes were incubated in the appropriate secondary antibodies diluted in 5% milk/PBS for 1 hour at room temperature. Membranes were again washed extensively in PBS before they were treated with enhanced chemiluminescence reagents (Perkin- Elmer, Boston, MA) as per the manufacturer's protocol and exposed to film (Clonex BioFlex MSIFilm,CLMS810).
11.10. Checking Protein Expression in Budding Yeast
Yeast cultures were grown overnight at 30°C in the appropriate selective media until a density of-0.5-1x10 cells/ml (OD600 -0.5-1.0). The preparation of yeast extracts used a modified protocol from Shire et al. (1999). Approximately 2xl07 cells were harvested, washed once in five pellet volumes of distilled water, and resuspended in three pellet volumes (-100 ul) of lysis buffer (25 mM Tris pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM DTT, and complete EDTA-free protease inhibitor cocktail [Roche Diagnostics, Cat. No.l 1873580001]) containing SDS sample buffer (25 mM Tris pH 6.8, 1% SDS, 0.1% bromophenol blue, 10% glycerol, ImM DTT). Glass beads (SIGMA, G8772) were carefully added to the mixture until it reached the meniscus of the lysis buffer. The mixture was vortexed vigorously for one minute, kept on ice for one minute, vortexed again, and finally boiled for 10 minutes. After a quick spin, 50 ul of the yeast whole cell extract was separated on 8% SDS-PAGE and western blot analysis was performed as described above.
53 III. RESULTS
III.l. hEBP2 is Able to Reconstitute EBV-Based Plasmid Maintenance in YPH499
In human cells, EBNA1 mediates the segregation of EBV-based plasmids by binding to the FR element and associating with host proteins on mitotic chromosomes. In budding yeast, EBNA1 is necessary but not sufficient to segregate plasmids containing the FR element. Plasmid maintenance can be increased efficiently by providing human EBP2, which tethers EBNA1 and EBV-based plasmids to mitotic chromosomes (Kapoor et ai, 2001). Using a similar yeast plasmid loss assay, I asked if the cellular proteins DEK, Brd2, MeCP2 or Brd4 can function like hEBP2 and confer maintenance stability to an FR-containing plasmid in actively dividing yeast cells in the presence of EBNA1. For the yeast plasmid loss assay (see Figure 6 for a schematic of the experimental protocol), yeast strain YPH499 was co-transformed to Ura, Leu and Trp prototrophy with three plasmids, respectively: a low-copy number plasmid expressing EBNA1 (or the empty vector control), a high-copy number plasmid expressing the potential mediator protein (or the empty vector control), and a high-copy number segregation test plasmid whose maintenance over several cellular generations will be closely monitored. The segregation test plasmids included pRS314, YRp7, and the FR-containing YRp7FR (see Figure 7A for a schematic of the segregation test plasmids). The transformants that grew on selective plates (SC-Ura,Leu,Trp) were inoculated and grown in selective media before they were transferred to media containing tryptophan (SC-Ura,Leu), which allows for the loss of the segregation test plasmids. Cells were grown for an additional 60 hours (or approximately 25 generations) before -200-300 colonies were spread onto selective versus non-selective plates. In parallel, 10-fold serial dilutions of the cultures were also spotted onto selective and non-selective plates. Plasmid stability was measured by taking a ratio of the number of colonies that grew on selective media compared to non-selective media. Two-tailed t-tests were performed by using the "True Positive" control (described below) as the condition to which all other test conditions are compared. The significance threshold was set at p<0.01. In addition to testing the ability of the candidate proteins to reconstitute YRp7FR plasmid maintenance in the presence of EBNA1, several controls were included in order to assess the ability of the candidate protein to maintain the FR plasmid in an EBNA1-dependent and FR-
54 TRP URA Test EBNA1 OPlasmi d LEU
\. J Candidate Protein
E ^ p««s«»g lf~~") lO
S(-UTL) S(-UTL) NS(-UL) Transformation Out-grow for -25 generations at 30°C
Calculate Plasmid stability: Spot and spread cultures
% colonies maintaining plasmid <^ ~J| f^^-cssa®^ fC^^^i = colonies on S/NS plates x 100 S(-UTL) NS(-UL)
Figure 6. The experimental protocol for the yeast plasmid loss assay. Yeast strain YPH499 was transformed with three plasmids: a segregation test plasmid (see Figure 7), an EBNA1- expressing plasmid (or its empty vector), and a high copy-number plasmid expressing the candidate protein of interest (or its empty vector). Transformants that grew on selective plates (S, SC-UTL) with respect to the segregation test plasmid were inoculated into selective media and grown overnight at 30°C. The next day, cultures were diluted and transferred to non-selective media (NS, SC-UL) with respect to the segregation test plasmid and grown for -25 generations at 30°C. After out-growth in NS media, 10-fold serial dilutions of the cultures were spotted on both S and NS plates. In parallel, -200-300 colonies were spread on separate S and NS plates. Plasmid stability (% Colonies Maintaining Plasmid) was measured by taking a ratio of the number of colonies found on S versus NS plates.
55 ARS1 /x» CEN6/ARSH4 ARS1 TRP1 TRP1 V/, TRP1
pRS314 YRp7 YRp7FR Autonomous Autonomous Autonomous Replication (ARSH4) (ARS1) (ARS1) Autonomous FR Element Segregation None (CEN6) (EBNA1-Dependent)
B. & Segregation Test & Controls (CTRLs) and Tests Plasmid <$? True Positive CTRL CEN/ARS - - FR-Specificity CTRL CEN/ARS - + TLST YRp7FR + + EBNA1 Alone CTRL YRp7FR + - Candidate Protein Alone CTRL YRp7FR - + FR Plasmid Alone CTRL YRp7FR - - FR-Specificity CTRL YRp7 - + True Negative CTRL YRp7 - -
Figure 7. Segregation test plasmids, controls, and test conditions used in the yeast plasmid loss assay. (A) The three segregation test plasmids used in the yeast plasmid loss assay each contains an ARS element that governs autonomous replication and a TRP1 selectable marker. pRS314 contains a CEN element that governs autonomous segregation and was used as the "True positive control" in the assay (see table in panel B). YRp7, which lacks any segregation element, was used as a "True negative control" for segregation. YRp7FR, which contains the EBV segregation element, FR, was the experimental test plasmid whose maintenance would depend on the presence of EBNA1. (B) Each candidate protein investigated in the yeast plasmid loss assay was tested for its ability to maintain YRp7FR in the presence of EBNA1 in order to address its role in the DNA segregation mechanism of EBV ("TEST" condition, shaded row). Maintenance was compared to the "True Positive" and "True Negative" controls, which monitor the stability of pRS314 and YRp7, respectively. In addition, maintenance of YRp7FR was investigated on its own, in the presence of EBNA1 but without a candidate protein, and in the presence of a candidate protein without EBNA1 to determine whether the candidate protein is functioning in conjunction with EBNA1. Finally, maintenance of pRS314 and YRp7 were also examined in the presence of the candidate protein to check for general effects on plasmid segregation. Plus "+" or minus "-" signs denote the presence or absence, respectively, of EBNA1 and candidate protein of interest in yeast.
56 specific fashion. The controls were as follows (see Figure 7B): The "True Positive" control tests the stability of the CEN/ARS-containing plasmid, pRS314. This plasmid can replicate and segregate autonomously; thus, it represents optimal plasmid maintenance ability in the YPH499 yeast strain. The "True Negative" control monitors the instability of YRp7. YRp7 can replicate autonomously due to the presence of the ARS1 element, but it is unstable and cannot partition equally to daughter cells since it lacks a centromeric (CEN) element. Thus, this control represents random or background segregation that occurs in YPH499. In addition, maintenance of YRp7FR was investigated on its own, in the presence of EBNA1 but without a candidate protein, and in the presence of a candidate protein without EBNA1 to determine whether the candidate protein was functioning in conjunction with EBNA1. Finally, maintenance of pRS314 and YRp7 were also examined in the presence of the candidate protein to check for general effects on plasmid segregation. In order to treat all samples identically, the empty p416MET25 plasmid, which does not encode EBNA1, and the empty p425PGK plasmid, which does not encode the candidate protein, were used in instances where EBNA1 expression and/or candidate protein expression were not desired. Previously, our laboratory used yeast strain KY320 for the plasmid loss assay studies (Kapoor et al, 2001). Here, I used yeast strain YPH499 as I noticed that it enabled higher transformation efficiencies, particularly when Brd2 and MeCP2 were introduced into yeast. In addition to this change, cultures were grown in non-selective media for -25 generations instead of 11 generations as previously done for KY320 because I observed a significant amount of background in the "True Negative" control after 11 generations of out-growth in YPH499. Since a new yeast strain was being tested in the plasmid loss assay, I first checked that hEBP2 was able to successfully reconstitute EBNA1-mediated segregation of the FR plasmid as observed previously. The results showed that, in the presence of both EBNA1 and hEBP2, 45% of the colonies were able to maintain the FR plasmid in an EBNA1-dependent and FR-specific fashion (Figure 8A). Although the efficiency with which hEBP2 and EBNA1 maintained the FR plasmid was statistically different from that of the "True Positive" control where 70% of the colonies were able to maintain pRS314 (p<0.01), FR plasmid maintenance was still ten-fold higher than in the absence of either EBNA1 or hEBP2. Therefore, hEBP2 could successfully reconstitute EBNA1-mediated segregation of EBV-based plasmids in YPH499. The yeast cultures were also harvested, lysed, and processed for Western blotting to analyze the protein
57 A. Segregation 3 es Test £ S % Colonies Maintaining Plasmid Plasmid w 9 NS (-UL) S (-UTL) 0 20 40 60 80 100
1 10-' 10-2 10-3 1 10-' 10-2 10-3 p-value < 0.01 B. EBNAl - + + hEBP2 - + + 100 i 70 55— 40 :hEBP2 «a 35 25 . —
55 «*mm I EBNAl 40 " I Actin
Figure 8. hEBP2 reconstitutes EBV-Based plasmid maintenance in YPH499. (A) The plasmid loss assay was conducted as shown in Figure 6 with hEBP2 as the candidate protein. Plasmid stabilities for all test conditions performed are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were performed relative to the "True positive control". Asterisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of YPH499 lysates expressing no EBNAl and no hEBP2 (lane 1) or EBNAl and hEBP2 (lanes 2 and 3 are lysates from two independent cultures). Blots were probed with anti-EBNAl, anti-hEBP2 and anti-actin antibodies. Arrow heads point to expected protein sizes. Asterisk (*) indicates non-specific protein bands or hEBP2 dimers.
58 stability of EBNA1 and hEBP2 in YPH499. Both EBNA1 and hEBP2 were expressed (Figure 8B). Thus, YPH499 was a suitable strain to use in my studies and I proceeded to test the candidate proteins DEK, Brd2, Brd4 and MeCP2.
111.2. DEK Does Not Promote FR Plasmid Maintenance in Budding Yeast
In contrast to hEBP2, DEK was unable to confer stability on YRp7FR even in the presence of EBNA1. Only 0.4% of the colonies harboured the FR plasmid after 25 generations of out-growth in non-selective media (Figure 9A). The DEK-expressing yeast cells did not exhibit any detectable growth defects as observed by light microscopy and by monitoring their doubling time (-2.3 hours per generation). A Western blot analysis confirmed that DEK, which has an apparent molecular weight of -55 kDa, was expressed in yeast cells at the expected size (Figure 9B). It is worth mentioning that the rabbit polyclonal DEK antibody recognizes non specific epitopes in yeast lysates. Nonetheless, a protein band that ran at around 55 kDa was observed only when yeast cells were transformed with the DEK-expressing plasmid. Therefore, DEK does not support faithful EBV partitioning in budding yeast.
111.3. Brd2 and MeCP2 are General Mediators of Plasmid Segregation
Brd2 was found to maintain YRp7FR in the presence of EBNA1 with an efficiency of 62%, which is comparable to that of the "True Positive" control (Figure 10A). Western blot analyses confirmed that Brd2, which has an apparent molecular mass of -130 kDa, and EBNA1 were both expressed in YPH499 (Figure 10B). Surprisingly, Brd2 also enabled 55% of the colonies to maintain YRp7FR in the absence of EBNA1, indicating that Brd2 was not functioning through EBNA1. To address if the observed maintenance was specific to the FR element of the segregation test plasmid, Brd2 was tested with YRp7. Brd2 maintained YRp7 (71%) to levels comparable to that of the "True Positive" control. In addition, Brd2 was also found to further increase the maintenance of pRS314 at statistically significant levels (88%). Therefore, Brd2 had a general stimulatory effect on plasmid maintenance. The results for MeCP2 were very similar to those for Brd2. In the presence of both EBNA1 and MeCP2, 52% of the colonies were able to maintain the FR plasmid (Figure 11 A). However, MeCP2 alone was also able to confer FR plasmid stability in 39% of the colonies.
59 A. Segregation r; Test £ M % Colonies Maintaining Plasmid Plasmid § 5 NS(-UL) S (-UTL) 0 20 40 60 80 100 N=43~ CEN/ARS -70 N-5~ — — —8( N=4~ 0.4* YRp7FR N=191-2.5 * N=6>3.4 * N=43|h5.' * YRp7 N=6>1.6* N=23 *3.1 * 1 2 3 2 3 l io io- io- l IO1 io- io- i ' p-value < 0.01 B. EBNAl + + + DEK - + ' +
70-
55— (DEK
40—I
35—I I 55- : EBNAl
40- [ Actin
Figure 9. DEK does not promote FR plasmid maintenance in YPH499. (A) The plasmid loss assay was conducted as shown in Figure 6 with DEK as the candidate protein. Plasmid stabilities for all test conditions performed are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were performed relative to the "True positive control". Asterisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of YPH499 lysates expressing EBNAl and no DEK (lane 1) or EBNAl and DEK (lanes 2 and 3 are lysates from two independent cultures). Blots were probed with anti-EBNAl, anti-DEK and anti-actin antibodies. Arrow heads point to expected protein sizes.
60 A.
% Colonies Maintaining Plasmid 0 20 40 60 80 100
YRp7FR
1 10-' 10-2 10-3 1 10' 10-2 10-3 * p-value < 0.01 B. EBNAl - + + Brd2 - + + 170— 130— #Bj I EBNAl I Actin Figure 10. Brd2 is a general mediator of plasmid maintenance. (A) The plasmid loss assay was conducted as shown in Figure 6 with Brd2 as the candidate protein. Plasmid stabilities for all test conditions performed are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were performed relative to the "True positive control". Asterisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of YPH499 lysates expressing no EBNAl and no Brd2 (lane 1) or EBNAl and Brd2 (lanes 2 and 3 are lysates from two independent cultures). Blots were probed with anti-EBNAl, anti-Brd2 and anti-actin antibodies. Arrow heads point to expected protein sizes. Asterisk (*) indicates possible degradation product of Brd2. 61 A. Segregation ^ g Test £ o % Colonies Maintaining Plasmid pq » NS (-UL) S (-UTL) 0 20 40 60 80 100 Plasmid w S CEN/ARS 1 lO"1 lO"2 lO"3 1 10-1 10-2 103 * p -value < 0.01 B. EBNAl - + + MeCP2 - + + 130— 100— ***** 40 35— -nf ^mm - Figure 11. MeCP2 is a general mediator of plasmid maintenance. (A) The plasmid loss assay was conducted as shown in Figure 6 with MeCP2 as the candidate protein. Plasmid stabilities for all test conditions performed are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were performed relative to the "True positive control". Asteisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of YPH499 lysates expressing no EBNAl and no MeCP2 (lane 1) or EBNAl and MeCP2 (lanes 2 and 3 are lysates from two independent cultures). Blots were probed with anti-EBNAl, anti-MeCP2 and anti-actin antibodies. Arrow heads point to expected protein sizes. 62 Like Brd2, MeCP2 also stabilized YRp7 and further increased the stability of pRS314 at statistically significant levels (from 70% to 90%, p<0.01). MeCP2 was confirmed to be expressed in YPH499 at the expected molecular mass of -100 kDa (Figure 1 IB). Therefore, like Brd2, MeCP2 can confer stability to a variety of plasmids in budding yeast. III.4. mBrd4 Confers FR Plasmid Stability in the Presence of EBNA1 Mouse Brd4 (mBrd4) and human Brd4 (hBrd4) are 93 % identical in sequence (see Figure 12 for the protein sequence alignment between mouse and human Brd4). While mBrd4 is 1400 amino acids long with a predicted molecular weight of -156 kDa, hBrd4 is 1362 amino acids long and -152 kDa in size. At the time the plasmid loss assays were being conducted, mBrd4 but not hBrd4 was readily available in a yeast expression vector. Moreover, it had just been demonstrated that mBrd4 was able to successfully reconstitute E2-mediated plasmid maintenance in budding yeast (Brannon et ah, 2005). Therefore, the mouse homologue of Brd4 was used in my yeast plasmid loss assay studies. The results showed that in the presence of mBrd4 and EBNA1, 63% of the colonies successfully maintained the FR plasmid at levels comparable to that of the True Positive control (Figure 13A). Moreover, much like for hEBP2, mBrd4-mediated segregation occurred in an EBNA1-dependent fashion since, in the presence of mBrd4 but absence of EBNA1, only 6% of the colonies were able to maintain the FR plasmid. In addition, Brd4 expression did not stimulate the segregation of YRp7 or pRS314. Therefore, the results suggested a role for Brd4 in EBNA1-mediated segregation of EBV plasmids. Western blots confirmed that EBNA1 was expressed in the yeast cells (Figure 13B) and a slight decrease in EBNA1 protein levels was seen in the presence of mBrd4 compared to in its absence. Nevertheless, the lower EBNA1 expression was sufficient for FR plasmid maintenance in the presence of mBrd4. The anti-Brd4 antibody detected a strong, non-specific protein band that ran at -130 kDa both in the presence and absence of mBrd4. Since full-length mBrd4 has a predicted molecular weight of -156 kDa, it is most likely behind this large band. However, a band that ran at -100 kDa was detected only in mBrd4-transformed yeast cells. This smaller protein band may represent a degradation product of the full-length mBrd4 protein. Based on this proposed degradation product, mBrd4 was likely expressed to similar levels in yeast cells with or without EBNA1. 63 bBri4/l-1362 1 SGPGTRLRNLPVMGDGLETSQMSTTQAQAQPQPANAASTNPPPPETSNPNKPKKQTNQLQYLLRVVLKTLWKHQFAWPFQQPVDAV 90 mBrttl-1400 1 SCPCTRLRNLPVMGDGLITSQMSITQAQAQPQPAXAASIN7?PPEISXPSKPKEQTNQLQVLLRVVLKIL\»'KHQrA\S'PFQQPVDAV 90 ffiri4/l-1362 91 KL>'LPDYYKIIKTPSIDMGTIKKSLEN_NYYWNAQECIQDFN'rMFT>XVIYNKPGDCIViMAIALIiCLFLQKIN'£LPIEEIEIMIVQAKGRG 180 BiBi'M-USO 91 KLNLPDYYKIIKTPMDMGIIKKRLINNYYWNAQECIQDFNIMFT^CYlYNKPGDClVLMAEALEKLFLQKIN'ELPTIETEIMn'QAKGRG 180 hBri4;:l-1362 181 RGRKETGIUCPGYSTYPNITQASIPPQTQTPdPKPPP i'QATftPFPAVTPDLl|vbl>VMT|vk'PPQPLQTPP>VPPQPBbPPAP&QPYQ 269 , , , :mBrti4/l-l«0 181 KGRKETGpKPGVSIVPNTTQASTyPQTQIPqgSPPPIP rQATpPFPAVTPDLI^I^YMT||7PQPLQIPg| Y?PQp|pj PPAp|vp QPYQ !"0 bBrf4/l-1362 270 SHPPIIA|A]rPQPVKTKKGTCRKADTTTPTTIBPIH£PPSl|plPEPKTto^ SEQIKCCSG 359 BiBrd4/i-i40o ::i SHPPn.^PQ?VKTKKGYKRKADTITPTTI0?lHEPPH|AjPEPKl|A]CLG|pjRRESSRPYKPPKKDVPDSQQHP|GJP£KSSK 5EQLKCCSG m bBri4/l-13tt M ILKEMFAKKHAAYAWPFYKPYm'IALGLHCYCDIIKHPhiDMSTIKSKLFJAkEYRDAQEFGADYRLMFSXCYKYNPPDHIVVAMARKLQDY 449 BBiMMM 361 ILKEMrAKKHAAYAWPrYKPVDVEALCLHDYCDIIKHPllDMSIIKSKLIgpEYRDAQEFGADVRLMFSXCYKYSPPDHIYYAJLARKLQDY 450 bBiit'1-1362 458 FEWFAKMPDE?EIPYV|Ah!SSPAVPPPTKVVAPPSSSDSSSl)SSSDSDSSTDDSEEERAQKLAELQEQLKAVHEQLAALSQPQQNK?KKK 539 mBiiM-1400 451 FFmFAOP0E?EEPVYJT]\?SSPAY?PPTKVVAPPSSSDSSSDSSSDSDSSTIiDSEEERAQRLAELQEQLKAyHIQLAAiSQ?QQNKPKKK 540 bBni4il-1362 54« EOKKFKKKEKH{&EEYEENKKSK&EiP?KKTKKXNSSXSNYSKKEl&fi&PPPTYESEEEDKCKPMSYEEKRQL.SLDINKLPCEK 629 oBrfW-1400 541 FKMKEKKKIKHii|pEEYEE>MSK|TjKEy??KKTKON%$NS^ 630 lBnl«-]M2 m LGRVVHIIQSREPSLKNSNPDEIElDFETLKPSTLRELERYVTSCLRKKRKPQAIKVBVIAGSSgMKGFSSSESESlslSESSSSDSIDSET •19 mUHUm 631 LGRVVHIIQSREPSLKNSXPMIEIDFETLKPSTLRELERYVTSCLRKKRKPQAEKYDYIAGSSKMKGFSSSESEs|lJsiSSSSDSEDSET bBtil4;l-1362 729 EMAP KS KKKGH|PT;R|E]QKKHHHM; )|APAPVPQQPPPPPQQPPPPPPPQQQQd-pPPPPPPPSMPQQEkPAMKSSPPPFIATbVPYL BtBtMl-1480 "21 E M AP KS KKKGHPR|DJQKKHHHHH: gAPAP\7QQPPPPPQQ??PPPPPQQQQ^PPPPPPPSM?Q^PA.\lKSSP?PFl|TApVPYL 810 bBriia-U« 809 EPQLPGSYFDPl(G|lFTQPILHiPQPELPPHLPQPPEHSIPPHLN"QHAYYSPPALHNAL?QQPSRPSNRAAALPPKPlAkPPAYSPAL 898 aBrd4/l-U00 811 EPQLPGSVFDPl|sfiFFQ?ILHLPQ?EL?PHiPQPPEHSIP?HiNQHAYYSPPALHNALPQQPSRPSNRAAALPPKP[ipPPAYSPAL 900 bBrd4/l-1362 89? LlPQPPMAQPPQYLLEDEEPPAPPlISMQMQLYLQQtQKYQPPTPLLPSYKVQSQPPPPLPPPPHPSYQQQpiQElQPPPPPPPQPQPPPQ 98" iiiBnM,l-1400 901 LLPQPPMAQ?PQYLLIDEEPPAPPLISMQMQLYLQQLQKVQPPTPLLPSVKYQSQPPPPLPPPPHPSV^^(Jg|QPPPPPPPQPQPPPQ 990 bBf#'l-13«2 98S QQHQPPPRPVHlI^&SJT^IQQPPPPJ^QQPJpTHPPPGQQPPPPQPAKFQQVlQHlftPRHHKSDPyslffePRHHKSSPYS|fbHLREAPSPUiIHSPqEIsbFQb 10" oBriH.1-1400 991 QQHQPPPRPVHI|P^FS^IQQPPPI^QQP|TJlPPPGQQPPPPQ?AKPQQVI^By5PRHHKSDPYslA^5PRHHKSDPYS|ApHLREAPSPLSIIHSP(^IPpFp Q 1080 hBrWl-Ue 1018 SLTHQSPPQQNYQPKtd - bEL[|AA|5YVQPQPLYYTKEEKIHSP 11RSEPF S^LRPEPP 1134 mBrM-1400 1081 SLTHOSPPOONVOPKiqOVKGRAEPOPPGPVMGOCOGCPPASPAAVPStlSPELBJPP 3VVOPOPLYVYEEEKIHSPIIRSEPFS F51RPEPP 11-0 lsBrA4 Figure 12. Protein sequence alignment of human and mouse Brd4. Brd4 sequences from human (hBrcW) and mouse (mBrd4) were aligned using Clustal method in Jalview. Boxed regions represent amino acids that are not identical among the Brd4 homologues. Human and mouse Brd4 share 93% protein sequence identity. 64 A. Segregation <; % Colonies Maintaining Plasmid Test g % 0 20 40 60 80 100 Plasmid W PP NS (-UL) S (-UTL) f\ ITS ^ % jj& £* *ft .» N=43_l -70 CEN/ARS N=6J[ 52 , i - • ;? * Q \J v# •**. N=20J -63 <' • N .5. .•> If* « N=19J * YRp7FR • h2.£ C- • -,*: *• G N=10l 1-6. 0* ('* , • ;:: wC € N=43J1-5.1 * I II N=10Jt-6 . 8* YRp7 1 ' €? O cl* -t €| ^ t» N=23) H3.1 * 1 101 10-2 10-3 1 101 10-2 103 * p-value < 0.01 B. EBNAl + + + - - - + + + Brd4 - - - + + + ++ + 170 — 130 — 100 — Jj| 55—I jj|< EBNAl B- Figure 13. mBrd4 reconstitutes EBV-Based plasmid maintenance in YPH499. (A) The plasmid loss assay was conducted as shown in Figure 6 with mBrd4 as the candidate protein. Plasmid stabilities for all test conditions performed are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were performed relative to the "True positive control". Asterisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of triplicate YPH499 lysates expressing EBNAl and no mBrd4 (lanes 1-3), mBrd4 and no EBNAl (lanes 4-6), or EBNAl and mBrd4 (lanes 7-9). Blots were probed with anti-EBNAl, anti-Brd4 and anti-actin antibodies. Arrow heads point to expected protein sizes. Full-length mBrd4 is likely masked by the large non-specific protein band that runs at -130 kDa. Asterisk (*) indicates possible degradation product of mBrd4. 65 III.5. The N-terminus of EBNA1 is Required for mBrd4-Mediated Segregation of the FR Plasmid The EBNA1 sequences that play a role in mitotic chromosome attachment and segregation in human cells were previously mapped to amino acids 8-67 and amino acids 325- 376, where deletion of residues 325-376 (EBNA1A325-376) had a major effect on both processes and deletion of residues 8-67 (EBNA1A8-67) had a minor effect on both processes (Wu et al, 2000; 2002; and see Figure 2 for the functional domains of EBNA1). These EBNA1 deletions were shown to have a similar effect on hEBP2-mediated segregation of FR plasmids in yeast (Kapoor et al, 2001; Wu et al, 2002). In order to determine if the Brd4 effect on plasmid stability involves EBNA1 sequences known to be important for EBV segregation in human cells, I investigated the ability of EBNA1 mutants to support partitioning of YRp7FR in yeast in conjunction with mBrd4. To this end, mBrd4-transformed cells were tested with three EBNA1 mutants (EBNA1A325-376, EBNA1A8-67 and EBNA1A61-83) in the yeast plasmid loss assay and their ability to maintain YRp7FR was compared to that of wild-type EBNA1. Of interest, EBNA1A61-83 was previously shown to support EBV-base plasmid segregation as well as replication in human cells but was defective in transcriptional activation (Wu et al, 2002). The results showed that EBNA1A325-376 consistently maintained YRp7FR in the presence of mBrd4 (Figure 14A). Moreover, while maintenance was slightly reduced compared to wild-type EBNA1 (49% versus 63%), this difference was not statistically significant (p>0.01). In contrast, EBNA1A8-67 or EBNA1A61-83 did not support the maintenance of YRp7FR in the presence of mBrd4. These results indicated that mBrd4-mediated segregation required the N- terminus of EBNA1. This observation was interesting since it contrasted with previous results which showed that residues 325-376 of EBNA1 were required for hEBP2-mediated segregation of YRp7FR (Kapoor et al, 2001). This strongly suggested that while EBNA1 is able to utilize both hEBP2 and mBRD4 to mediate FR plasmid maintenance in yeast, the mechanism by which EBNA1 performs these processes differs. This is in keeping with the hypothesis that EBNA1- mediated segregation involves multiple protein partners and various mechanistic pathways. To ensure that all EBNA1 mutants were expressed at levels similar to that of wild-type EBNA1, triplicate independent cultures (labelled I, II and III in Figure 14B) from each test condition of the plasmid loss assay were harvested and processed for Western blot analyses. 66 A. Segregation < Test % Colonies Maintaining Plasmid PQ 3 Plasmid W £ NS (-UL) S (-UTL) 0 20 40 60 80 100 CEN/ARS <§ H N=43 -70 WT + N=20 -63 A325-376 + G O # & -J*. N=10 -4 B. EBNAl WT A325-376 A8-67 A61-83 I II III I II III I II III I II III EBNA1 Actin 12-3 45 67 8 9 10 11 12 % colonies v? s? v? ^ 0\ t> VO ft in N O^ O^ 0s 00 maintaining r- in oo >n en t- >n O ro I-H c4 o\ in r-< plasmid Figure 14. The N-terminus of EBNAl is required for mBrd4-mediated segregation of the FR plasmid in yeast. (A) EBNAl mutants lacking amino acids 325-376 (EBNA1A325-376), 8-67 (EBNAl A8-67) or 61-83 (EBNA1A61-83) were tested in the plasmid loss assay as shown in the schematic of Figure 6 with mBrd4 as the candidate protein. Plasmid stabilities are shown in the provided bar-graph. Error bars indicate standard deviation. Two-tailed t-tests were conducted using wild-type EBNAl as the sample to which all other conditions were compared. Asterisks (*) indicate statistical significance with a p-value <0.01. N-values indicate the number of trials performed for each test condition. (B) Western blot of YPH499 lysates from three independent cultures (I, II, and III) expressing mBrd4 and either wild-type EBNAl (lanes 1-3), EBNAl A325-376 (lanes 4-6), EBNA1A8-67 (lanes 7-9) or EBNA1A61-83 (lanes 10-12). Blots were probed with anti-EBNAl and anti-actin antibodies. The plasmid stability (% colonies maintaining plasmid) that was calculated for each culture is shown. 67 These cultures were spotted and spread on both selective and non-selective plates earlier in the day; thus, it was possible to measure plasmid stability for each of the cultures examined. The results showed that wild-type EBNA1 was expressed at variable levels in YPH499. Nevertheless, greater than 59% of the colonies from each culture could still successfully maintain the FR plasmid (Figure 14B, lanes 1-3). Surprisingly, the yeast culture that expressed the highest level of EBNA1 was also the least able to mediate FR plasmid segregation (compare lane 2 versus lanes 1 and 3). Yeast cells expressed EBNA1A325-376 at levels comparable to or higher than wild-type EBNA1 although plasmid maintenance ability was slightly hindered for the 325-376 deletion mutant (compare lanes 4-6 versus lanes 1-3). Intriguingly, a similar pattern was observed here with wild-type EBNA1: yeast cultures expressing the highest level of EBNA1 were also the least able to maintain the FR plasmid (compare lane 5 versus lanes 4 and 6). EBNA1A8-67 (lanes 7-9) and EBNA1A61-83 (lanes 10-12) were expressed at levels comparable to and in some cases higher than wild-type EBNA1; however, these proteins were unable to consistently maintain YRp7FR. It is important to point out that while EBNA1A8-67 had consistently been shown to be defective in maintaining the FR plasmid, in one case here (lane 7), 55% of yeast colonies showed positive activity. This was an outlier from the data set and was not reproducible. Taken together, my results suggest that the N-terminal domain of EBNA1 is required for mBrd4-mediated segregation. III.6. The Transactivation Domain of EBNA1 Mediates an Interaction between EBNA1 and hBrd4 To determine if EBNA1 and Brd4 interact in human cells and also to map the domain of EBNA1 required for this association, 293T cells were co-transfected with an EBNA1-expressing plasmid and a plasmid encoding human Brd4. The EBNA1 mutants used in this co- immunoprecipitation (co-IP) assay are shown in Figure 15 and include the following: wild-type EBNA1, EBNA1A325-376, EBNA1A8-67, EBNA1A8-67/325-376, EBNA1A61-83, and the C- terminal fragment of EBNA1 spanning amino acids 452-641, which contains the DNA binding and dimerization region (DBDR) and served as the negative control in this assay. Cells were harvested 3 days post-transfection, and lysates were treated with DNasel prior to immunoprecipitation with Brd4 antibodies and protein A-agarose. To determine the degree of 68 DNA-binding and Figure 15. Schematic diagram of EBNA1 mutants tested in the co-immunoprecipitation (co-IP) experiments. All EBNA1 deletion mutants were constructed from EBNA1 that lacked the Gly-Ala repeat. This was considered as wild-type EBNA1 in my studies. EBNA1 mutants used in the co-IP experiment have deletions in the following amino acids: 8 to 67 (EBNA1A8- 67), 325 to376 (EBNA1A325-376), 8 to 67 and 325 to 376 (EBNA1A8-67/325-376), and 61 to 83 (EBNA1A61-83). Moreover, the C-terminal fragment of EBNA1 (amino acids 452-641) which contains the DNA-binding and dimerization region (DBDR) was used as the negative control in these assays. All EBNA1 mutants contained a nuclear localization signal (NLS). 69 background from non-specific binding of EBNA1 and EBNA1 mutants to the agarose beads, each lysate was also incubated with the beads in the absence of the Brd4 antibody (odd- numbered lanes labelled "C" stands for "Control" in Figure 16). Immunoprecipitated proteins were examined by SDS-polyacrylamide gels and Western blotting using antibodies against the DBDR of EBNA1 and Brd4. Moreover, 5% of the total amount of input protein used in each immunoprecipitation reaction was also examined by SDS-polyacrylamide gels and Western blotting to assess the expression of Brd4 and EBNA1. The results from two separate experiments are shown in Figure 16 (top and bottom panels are duplicate experiments). A striking feature about Brd4 is that while it has a predicted molecular weight of 156 kDa, it has an apparent weight of -200 kDa in SDS-polyacrylamide gels independent of the cell line in which it was examined (Dey et al, 2000; You et al, 2006). This is consistent with my results where Brd4 was running above the 170 kDa marker (see Figure 16, Input lanes 1-6). Moreover, this band was expressed at least 10-fold higher compared to cells transfected with the empty vector control (data not shown), suggesting that Brd4 was efficiently over-expressed in 293T cells. The co-IP results indicated that wild-type EBNA1, but not the 452-641 EBNA1 fragment, was able to interact with Brd4 (Figure 16, lanes 7-8 and 17-18). Moreover, consistent with the yeast plasmid loss assay, EBNA1A325-376 bound Brd4 (lanes 9-10) to a similar degree as that of the wild-type protein. Surprisingly, EBNA1A8-67 and the double-deletion mutant EBNA1A8-67/325-376 both interacted strongly with Brd4 (lanes 11-14), even though these EBNA1 mutants were expressed at lower levels compared to wild-type EBNA1 and EBNA1A325-376 (compare Input lanes 3-4 versus lanes 1-2). This observation appears inconsistent with the plasmid loss assay since EBNA1A8-67 cannot reconstitute FR plasmid maintenance (Figure 14). Nevertheless, taken together, the data suggest that increased binding of EBNA1 to Brd4 might be inhibitory to segregation while a transient interaction is likely optimal for segregation (see Discussion Section IV.3.). In contrast to the results observed for EBNA1A8- 67, EBNA1A61-83 was unable to bind Brd4 even though it was well-expressed (Figure 16 lanes 5 and 15-16). This correlates well with the inability of EBNA1A61-83 to confer FR plasmid stability in yeast cells. In summary, the results presented here indicate that residues 67-83 of EBNA1 form a minimal binding region with Brd4. Moreover, deletion of residues 8-67 may expose this site and account for the observed increase in binding to Brd4. 70 1/20 Input IP: a-Brd4 cn c- cn i-H en -37 6 t^ 7/32 J v© 55 . .a _ < 001 ^o 1—1 < , ' t- C- 00 Qb «N z,Ui en 00 00 VO >/•> W Brd4 no----r— no- *•§**« 130- _J_ 130_ -m " 55 *« ^ 55—• 4ft •fjji 1/20 Input IP:a-Brd4 VO ec-n- • v© i t- ID rj en e'>o i—i en en £ s < t-~ 7/3 : 00 v© 3 Pi «2 en ^ 1 8- 6 rn 8- 6 v© •7- V» V© ^D ' i W < < < < •G SP3 e.An o&o oto v°o? «^ W < < < < •* C B C B C B C B C B C B oak Figure 16. EBNA1 binds Brd4 and EBNA1 residues 61-83 are required for the interaction. Top and bottom panels are the results obtained from two independent experiments. 293T lysates from cells co-expressing Brd4 and either EBNA1, EBNA1A8-67, EBNA1A325-376, EBNA1A8-67/325-376, EBNA1A61-83 or 452-641 were digested with DNasel, pre-cleared with protein-A agarose and mixed with protein-A agarose beads overnight at 4°C with (B) or without (C) anti-Brd4 antibodies. Beads were spun down, washed, and eluted with SDS. Eluted protein complexes (right, lanes 7-18) and 1/20 of input lysates (left, lanes 1-6) were analyzed by Western blotting with anti-Brd4 and anti-EBNAl antibodies. 71 III.7. EBNA1 Remains Associated with Mitotic Chromosomes Upon Brd4 Down- Regulation Our laboratory previously showed that silencing hEBP2 in human cells resulted in the dissociation of EBNA1 from mitotic chromosomes as observed by indirect immunofluorescence microscopy of metaphase spreads and by biochemical fractionation of mitotic cells into soluble and chromatin-associated fractions (Kapoor et al, 2005). In the latter case, hEBP2 silencing resulted in the shift of more than 50% of EBNA1 from the chromosomal pellet fraction to the soluble fraction. These results strongly supported the contention that hEBP2 is essential in tethering EBNA1 to cellular DNA during mitosis, thereby facilitating EBV episome partitioning. In order to determine if Brd4 plays a similar role in EBNA1 attachment to mitotic chromosomes, I performed a similar biochemical fractionation experiment using mitotic cells down-regulated in Brd4 protein expression. Moreover, I was also interested in assessing the effect of silencing Brd4 on EBNA1 attachment to condensed chromatin during G2. It is hypothesized that EBNA1 is loaded onto chromosomes during G2 since immunofluorescence microscopy experiments performed in our laboratory showed that EBNA1 was already associated with DNA during early prophase (Nayyar and Frappier, unpublished data). Thus, investigating EBNA1 localization during G2 upon Brd4 silencing would address the role of Brd4 in loading EBNA1 onto condensed DNA. For these experiments, I generated endoribunuclease-prepared short interfering ribonucleic acids (esiRNAs) appropriate for silencing Brd4 in HeLa cells (henceforth referred to as esiBRD4). In brief, a 392 bp region of Brd4 that spans nucleotides 153-508 was selected by the DEQOR software program as the region that contained optimal silencing potential with least amount of non-specific off-target effects. This region of Brd4 was PCR amplified, in vitro transcribed, annealed to form double-stranded RNA products, digested with RNAselll into 18-21 bp fragments, and finally purified by ethanol precipitation. HeLa cells transiently expressing EBNA1 from an EBV-based plasmid containing oriP were transfected with esiBRD4 or small interfering RNA (siRNA) against GFP (siGFP) as a negative control. To check for Brd4 silencing efficiency, a small proportion (-10%) of the cells harvested for biochemical fractionation analyses were lysed and resolved by SDS-PAGE separately. Optimal silencing was achieved three days post transfection. Of interest, HeLa cells which had been treated with 72 esiBRD4 did not show obvious growth defects when compared to siGFP-treated cells, since both sets of cells reached similar confluence levels three days post-transfection. Biochemical fractionation was performed as described in Materials and Methods in Section II.7. In brief, Brd4 knock-down cells blocked in mitosis with colcemid were harvested by mitotic shake-off. In addition, the cells that remained attached to the plate after mitotic shake-off were harvested separately as these cells were previously shown to be in the G2 phase of the cell cycle (Sakwe et al., 2007). Whole cell (W) lysates were then generated and fractionated into soluble (S) and chromosomal pellet (P) fractions by low-speed centrifugation. Equal cell equivalents of each fraction (W, S, and P) were analyzed by Western blotting with an antibody specific to EBNA1. The fractions were also probed with several other antibodies against previously characterized proteins to confirm that the fractionation procedure was successful and that these proteins localized to the expected fractions. For G2, nucleosome assembly protein 1 (Napl) served as a soluble protein control while Brd4, hEBP2 and Mcm2 (minichromosome maintenance complex component 2) served as markers for the chromosomal pellet. For mitosis, Mcm2 and Napl served as soluble protein controls while Brd4 and hEBP2 served as markers for the chromosomal pellet. The results from two independent experiments are shown in Figure 17 (top and bottom panels are duplicate experiments). In both experiments, Brd4 was efficiently down-regulated when compared to siGFP-treated cells (Figure 17A and C). Surprisingly, EBNA1 protein levels appeared to be up-regulated upon Brd4 silencing; however, this effect was not always seen and did not affect the fractionation results (data not shown). In G2, EBNA1, Brd4, hEBP2 and Mcm2 were predominantly chromatin-bound while Napl was soluble (Figure 17B and D, left panels), as expected. EBNA1 remained in the chromatin pellet upon silencing Brd4, suggesting that Brd4 does not play a major role in loading EBNA1 onto condensed chromatin. The localization of Napl and hEBP2 did not change. In mitosis, EBNA1 localized to the chromatin pellet fraction, as expected (Figure 17B and D, right panels). Upon knock-down of Brd4, EBNA1 localization was unaltered, suggesting that Brd4 does not play a major role in mediating EBNA1 attachment to mitotic chromosomes. hEBP2 and Mcm2 localizations were also unaffected in the absence of Brd4. Although the yeast plasmid loss assay data suggested that Brd4 plays a role in EBV segregation (Figure 13), the lack of dependence on Brd4 for EBNA1 binding to human mitotic chromosomes was not entirely surprising. Indeed, given that 73 A. B. G2 Mitosis g § siGFP esiBRD4 siGFP esiBRD4 •55 '8 WSPWSPWSPWSP Brd4 -no Brd4 -«# — «» -no «. _130 - —130 EBNA1 , EBNA1 • -*_„ hEBP2 Actin •* "*"«"• Napl Mcm2 —•*" ~=r-mm«mm -wgi ««*«••• C. D. G2 Mitosis £ g siGFP esiBRD4 siGFP esiBRD4 oa g- WSPWSPWSPWSP Brd4 *"~ _20° Brd4 M« , «m * . —no —170 ^ •- Figure 17. Silencing Brd4 does not affect EBNA1 fractionation with chromatin in G2 or mitosis. Top and bottom panels are the results obtained from two independent experiments. HeLa cells containing an EBNA1-expression plasmid were transfected with esiBRD4 or siGFP as control. Transfected cells were blocked in mitosis and equal numbers of cells arrested in mitotis (harvested by mitotic shake-off) or G2 (cells that remain attached to plates) were used to prepare whole-cell lysates (W). A fraction of the whole-cell lysates (50 ug) was analyzed by Western blotting with anti-Brd4, anti-EBNAl and anti-actin antibodies to assess silencing efficiency (A and C). The remainder of the whole-cell lysates were separated into chromosomal pellet (P) and soluble (S) fractions by low-speed centrifiguation using a biochemical fractionation assay as described in materials and methods. Equal cell equivalents from each fraction (W, S and P) were analyzed by Western blotting with anti-Brd4, anti-EBNAl, anti-hEBP2, anti-Napl and anti-Mcm2 antibodies (B and D). 74 EBNA1A61-83 failed to bind Brd4 (Figure 16) yet this mutant had no obvious defects in chromosome attachment or segregation function (Wu et al, 2002), it was expected that EBNA1 association with mitotic chromosomes would not depend primarily on Brd4. Nevertheless, this new data does not entirely contradict the results obtained from the yeast plasmid loss assay since at this point, it cannot be ruled out that the role of Brd4 in EBV segregation in human cells is minor (see Discussion Section IV.4.). Several interesting observations were made during the course of the biochemical fractionation experiments (see Figure 17B and D). First, although Brd4 has a predicted molecular mass of ~ 152 kDa, its apparent molecular mass during interphase was -200 kDa and during mitosis, it was -170 kDa. This suggested that Brd4 may be differentially modified at different stages of the cell cycle. However, further analyses of the physiological properties and functions of Brd4 were beyond the scope of this thesis. The second interesting observation was that Brd4 silencing altered the localization of other cellular proteins throughout the cell cycle. The fraction of Nap 1 that localized to the chromosomal pellet in mitosis was found to increase upon Brd4 silencing. Moreover, Mcm2 was partially released from the chromatin and entered the soluble fraction when Brd4 was down-regulated during G2. However, the significance of these effects is not clear and it is not known if these effects are EBNA1 -dependent or independent. III.8. EBNA1 Remains Associated with Interphase Chromatin Upon Brd4 Down- Regulation While it is a well-known fact that EBNA1 is found in the nucleus during interphase, the literature is still conflicting as to whether EBNA1 is chromatin-bound or soluble during this phase of the cell cycle. While some have suggested that EBNA1 is soluble (Daikoku et al, 2004), others have shown that EBNA1 is associated with DNA (Ito et al, 2002; Kanda et al, 2001; Kanda et al, 2007; Nanbo et al, 2007; see Section 1.2.3). Using the biochemical fractionation protocol described in Materials and Methods Section H.7., I found that EBNA1 localized to the pellet fraction during G2 and that this occurred in a Brd4-independent fashion (Figure 17B and D; also see above). However, to assess the association of EBNA1 with DNA during interphase, I fractionated asynchronously-growing HeLa cells transiently expressing 75 EBNA1 on a plasmid. It is important to mention that asynchronously-growing HeLa cells harvested for these analyses contained less than 5% mitotic cells. For HeLa cells treated with siGFP, I found that EBNA1 localized to the pellet fraction in cells undergoing logarithmic growth (Figure 18 top and bottom panels are duplicate experiments). Moreover, such localization was unaffected upon treating cells with esiBRD4. Therefore, these results showed that EBNA1 does associate with interphase chromatin and furthermore, this is accomplished in a Brd4-independent manner. III.9. EBNA1 Remains Associated with Cellular Chromatin Upon Brd2 and MeCP2 Down- Regulation The yeast plasmid loss assay showed that Brd2 and MeCP2 were able to confer plasmid stability in the presence or absence of EBNA1 regardless of the segregation plasmid being tested (see Section III.3). This suggested that both Brd2 and MeCP2 were general mediators of plasmid maintenance in budding yeast. Nevertheless, since Brd2 and MeCP2 were able to maintain the FR plasmid in the presence of EBNA1,1 wanted to test if the mechanism by which this occurred was through EBNA1-mediated tethering onto cellular chromosomes. To this end, HeLa cells expressing EBNA1 were treated with siRNAs for Brd2 (siBRD2), MeCP2 (siMeCP2) or siGFP and processed in the biochemical fractionation protocol as described above. I assessed EBNA1 localization in cells undergoing logarithmic growth as well as cells in G2 and mitosis. Of interest, there were no obvious growth defects in siBRD2- or siMeCP2-treated cells when compared to siGFP-treated cells. To assess the silencing efficiency, a fraction of the asynchronously-growing and G2/M- blocked cells that were used in the biochemical fractionation experiments were harvested separately and analyzed by Western blotting. While Brd2 was efficiently silenced, MeCP2 was poorly silenced although some down-regulation of the protein was seen (Figure 19A). The fractionation experiments showed no effect of Brd2 or MeCP2 down-regulation on EBNA1 localization in asynchronously-growing cells, G2 or mitosis (Figure 19B, left panel). hEBP2 localization was also unaffected (Figure 19B, right panel). Therefore, Brd2 and MeCP2 do not play significant roles in mediating EBNA1 association with DNA regardless of the cell-cycle phase. 76 A. B. a Log siGFP esiBRD4 esiBR I siGF P W S P W S P Brd4 - Brd4 -«*ai —— iBNAl —_ — » EBNA1 «. — _ — Actin • „ •• hEBP2 m *m—m mm C. D. Log £ 3 siGFP esiBRD4 .9 -a • W S P W S F CO U EBNA1 — —•— — Figure 18. Silencing Brd4 does not affect EBNA1 fractionation with chromatin during logarithmic growth. Top and bottom panels are the results obtained from two independent experiments. HeLa cells containing an EBNA1 -expression plasmid were transfected with esiBRD4 or siGFP as control. Transfected cells in logarithmic growth were used to prepare whole-cell lysates (W). A fraction of the whole-cell lysates (50 jig) was analyzed by Western blotting with anti-Brd4, anti-EBNAl and anti-actin antibodies to assess silencing efficiency (A and C). The remainder of the whole-cell lysates were separated into chromosomal pellet (P) and soluble (S) fractions by low-speed centrifiguation using a biochemical fractionation assay as described in materials and methods. Equal cell equivalents from each fraction (W, S and P) were analyzed by Western blotting with anti-Brd4, anti-EBNAl and anti-hEBP2 antibodies (B and D). 77 A. Log G2/Mitosis CN CN p~* ft VI llllillm MeCP2 |iii|l|ij|§^ 4| EBNA1 ~"^T^| «j| B. siGFP siBRD2 siMeCP2 siGFP siBRD2 siMeCP2 G2 Mitosis WB: EBNA1 WB: hEBP2 Figure 19. Silencing Brd2 or MeCP2 does not affect EBNA1 fractionation with chromatin throughout the cell-cycle. HeLa cells containing an EBNA1-expression plasmid were transfected with siBRD2, siMeCP2 or siGFP as control. Transfected cells were blocked in mitosis as required. Whole-cell lysates (W) were prepared from equal numbers of cells in logarithmic growth or in mitosis/G2. A fraction of the whole-cell lysates (50 ug) was analyzed by Western blotting with anti-Brd2, anti-MeCP2, anti-EBNAl and anti-actin antibodies to assess silencing efficiency (A). The remainder of the whole-cell lysates were separated into chromosomal pellet (P) and soluble (S) fractions by low-speed centrifiguation using a biochemical fractionation assay as described in materials and methods. Equal cell equivalents from each fraction (W, S and P) were analyzed by Western blotting (WB) with anti-EBNAl and anti-hEBP2 antibodies (B). 78 IV. DISCUSSION The importance of using the yeast plasmid loss assay for initial investigations is two fold: first, it allows for the development of an isolated system to specifically test cellular proteins for their role in conferring stability to the normally unstable EBV-based plasmid. Second, by using budding yeast as the model organism, the replication and segregation activities of EBNA1 can be separated, thus allowing for the sole study of EBNA1-dependent segregation. hEBP2 was the first cellular protein tested to successfully reconstitute FR plasmid maintenance in yeast in an EBNA1 -dependent manner (Kapoor et ah, 2001). Using a different yeast strain, YPH499,1 showed that hEBP2 was able to maintain the FR plasmid even after 25 generations of growth in non-selective conditions with respect to the test plasmid (Figure 8). This confirmed previous results that hEBP2-mediated segregation in budding yeast is not strain-specific. Moreover, I was able to use this new yeast strain, which offers high-efficiency transformations, to test if the cellular proteins DEK, Brd2, MeCP2 and Brd4 could function like hEBP2 in conferring stability to the FR plasmid in budding yeast. IV.l. DEK is Unable to Mediate FR Plasmid Maintenance in Yeast The human DEK proto-oncogene was originally identified as a fusion with the CAN nucleoporin protein in a subset of patients with acute myeloid leukemia (AML) (von Lindern et ah, 1990). DEK is a DNA-binding protein that associates with chromatin during both interphase and mitosis (Kappes et ah, 2001). It is an abundant nuclear protein that is found in all multicellular organisms (animals, plants and fungi) but it is not found in yeast (Waldmann et ah, 2004 and references therein). As described earlier (see Section 1.3.2.), DEK was found to interact and co-localize with KSHV LANA1 on mitotic chromosomes (Krithivas et ah, 2002); thus, it has been implicated in KSHV episome persistence although no functional studies have been conducted to prove this. For these reasons, DEK was a good candidate protein to test in the plasmid loss assay. The results presented here clearly show that DEK is unable to confer maintenance stability to the FR plasmid even in the presence of EBNA1 (Figure 9). Further statistical analyses reveal that FR plasmid maintenance in the presence of DEK and EBNA1 (0.4%) is not considered statistically different from the EBNA1 alone control (2.5%, p=0.33), the DEK alone 79 control (5.2%, p=0.012), or the FR plasmid alone control (5.1%, p=0.014). Nevertheless, the presence of both DEK and EBNA1 reduced FR plasmid maintenance by 6 to 12-fold compared to DEK alone, EBNA1 alone, or the FR plasmid alone. ARS-containing plasmids undergo many rounds of replication per cell cycle and are present at high-copy numbers in cells. However, as ARS-containing plasmids lack segregation elements, they are randomly distributed between cells and are therefore unstable. My results suggest that the combined functions of DEK and EBNA1 may slightly inhibit the random partitioning of the FR plasmid. EBNA1 binding to the FR element of the FR plasmid may form large clusters of protein-DNA complexes in vivo, as has been described in vitro (Frappier and O'Donnell, 1991; Middleton and Sugden, 1992). An increase in plasmid instability could occur if these clusters were biased to remain within either the parent cell or the newly-formed daughter cell. DEK has been shown to bind circular plasmid DNAs that have been reconstituted with histone octamers and induce positive supercoils which severely inhibits replication (Alexiadis et al, 2000; Waldmann et al, 2002). It is therefore possible that the combination of DEK, which may inhibit FR plasmid replication, and EBNA1, which could inhibit equal partitioning of plasmids, results in low FR plasmid maintenance levels as observed here. Wise-Draper and colleagues recently showed that over-expressing DEK in HeLa cells inhibited apoptosis by interfering with p53 functions (Wise-Draper et al, 2006). Moreover, they found that knock-down of DEK in HeLa cells led to cell death, implicating DEK as a protein involved in cellular survival. As such, testing the effect of DEK silencing on EBNA1 attachment to cellular DNA would have been challenging and was therefore excluded from my studies. Nevertheless, based on the yeast plasmid loss assay, it is unlikely that DEK plays a role in EBV segregation in human cells although this possibility cannot be completely excluded at this point. IV.2. Brd2 and MeCP2 are General Mediators of Plasmid Maintenance Brd2 and MeCP2 have both been implicated in KSHV segregation based first on their ability to interact with LANA1, and second by immunofluorescence microscopy studies which showed their co-localization with LANA1 on host mitotic chromosomes (Piatt et al, 1999; Viejo-Borbolla et al, 2005; Mattsson et al, 2002; You et al, 2006; Krithivas et al, 2002). MeCP2 has also been proposed to mediate HVS episome persistence since it interacts with ORF73 and silencing MeCP2 caused HVS-based plasmid loss in mammalian cells (Griffiths and 80 Whitehouse, 2007; see Section 1.3.2.). Brd2 preferentially binds acetylated lysine 12 of histone H4 and this interaction occurs during both interphase and mitosis (Dey et al, 2000; Kanno et al, 2004; Dey et al, 2003). MeCP2 has also been shown to bind mitotic chromosomes (Krithivas et al, 2002; Griffiths and Whitehouse, 2007). Taken together, these observations suggested that both Brd2 and MeCP2 could be good candidates for governing EBV segregation. However, plasmid loss assays conducted in my studies revealed surprising results. Brd2 and MeCP2 were both able to confer maintenance stability on any plasmid that they were given. Even YRp7FR, whose maintenance is usually EBNA1 -dependent, was stable in the sole presence of either Brd2 or MeCP2 (Figures 10 and 11). Indeed, the presence of EBNA1 did not confer any statistically significant increase in the stability of YRp7FR compared to in its absence (p>0.01). The mechanism by which Brd2 and MeCP2 are able to mediate general plasmid maintenance remains elusive. Nonetheless, it is possible to make certain conjectures based on published data regarding the functions and properties of these two proteins. Brd2 is a short family member of BET proteins that contains two bromodomains and an ET domain but lacks the additional 650-amino acid C-terminal "tail" present in Brd4 (reviewed in Florence and Faller, 2001). It was originally identified as an autophosphorylating nuclear protein kinase whose activity is induced upon treatment of cells with the immunoregulatory hormome interleukin-la (IL-la) (Rachie et al, 1993; Denis and Green, 1996). Subsequent studies showed that Brd2 is involved in the transactivation of several cell-cycle regulatory genes, including cyclin Dl, cyclin 1 and cyclin E, implicating it as a regulator of Gl-S progression (Denis et al, 2000). Indeed, over-expression of Brd2 accelerates the cell cycle (Sinha et al, 2005). The transactivation function of Brd2 involves the ability of Brd2 to interact with acetylated histones via its bromodomain (Kanno et al., 2004) and to associate with multi-protein complexes involved in transcription and chromatin remodelling via its ET domain (Denis et al, 2006 and references therein). Brd2 also contains intrinsic kinase activity at its C-terminus and it is able to phosphorylate E2F-1, a component of the transcriptional complex to which it binds (Sinha et al, 2005). Co-immunoprecipitation experiments using differentially-tagged Brd2 proteins showed that Brd2 forms homodimers in vivo (Nakamura et al, 2007). Crystallization studies revealed that the first bromodomain of Brd2 mediates the dimerization and the interface that is formed contains two acetyllysine-binding pockets and a negatively charged secondary binding pocket, which may mediate sequence-specific contacts with the acetylated tail of H4 81 (Nakamura et al, 2007). Subsequently, it was shown that the dimerization is a prerequisite to interaction with acetylated tails of histone H4 (Nakamura et al, 2007). There are several possibilities to explain how Brd2 might ensure stable plasmid partitioning in cells: First, the ability of Brd2 to dimerize raises the possibility that while one monomer interacts with proteins on the host chromosomes, the other monomer might bind the plasmid (possibly through the second bromodomain), thereby tethering it to mitotic chromosomes. The interaction with the plasmid could be through histone interactions which would be expected to assemble on the replicating plasmids. Second, Brd2 might phosphorylate a protein in yeast cells, which then serves to govern stable plasmid partitioning either by serving as a mediator protein that tethers plasmids to mitotic chromosomes, by increasing plasmid replication and therefore plasmid copy-number, or by another mechanism. Third, since Brd2 is able to recruit chromatin remodelling complexes such as S WI/SNF to DNA (Denis et al., 2006), it is possible that the topology of the DNA plasmid is altered such that it has increased binding to Brd2 and increased stability. It is worth mentioning that over-expression of Brd2 in yeast cells might interfere with the functions of Bdflp, the yeast homologue of Brd2, which is involved in normal vegetative growth (Lygerou et al, 1994). Presumably, expression of Brd2 might lead to slow growth phenotypes and as a result, reduced plasmid loss rates. However, since no observable growth defects were seen when Brd2 was over-expressed, this is not a likely explanation. Two additional points needs to be addressed concerning Brd2-mediated plasmid segregation. First, when Brd2 was provided with YRp7FR, there was a high degree of variability in plasmid stability that varied 4.5-fold (from 20% to 90% of colonies were able to maintain YRp7FR, see Figure 10). This was not seen for pRS314 (77% to 97%) or for YRp7 (38% to 94%). The yeast assay system as a whole does contain some level of variability. This is likely due to protein degradation of Brd2 and/or EBNA1 which leads to variable levels of full-length protein, as well as to the fact that each cell may harbour different copy numbers of YRp7FR and 2-|am plasmids encoding the candidate protein of interest. However, these reasons alone are not sufficient to explain why the presence of FR in YRp7 causes instability in the function of Brd2- mediated plasmid segregation. It is possible that Brd2 has lower affinity for AT-rich sequences like the FR (Karlin, 1986), or FR confers a change in plasmid DNA topology that results in reduced affinity of the protein for the plasmid. However, neither observation has been 82 documented in the literature. Nonetheless, if true, cells that express higher levels of full-length Brd2 could have more plasmids bound by the protein and plasmid instability can be overcome. Thus, the large range in the observed plasmid maintenance can be accounted for. The second point that should be addressed is that Brd2 has been investigated in E2-mediated segregation in a similar yeast system by an independent group (Brannon et ah, 2005). Unlike in my studies, Brd2 was not shown to promote an increase in general plasmid stability (Brannon et ah, 2005). Indeed, Brd2 was unable to maintain the BPVl-based plasmid either in the presence or absence of E2. This could simply be due to the fact that Brd2 was expressed under the ADH1 promoter in their case instead of the PGK promoter as used in my yeast assay. However, as they did not address if Brd2 was efficiently expressed at the expected size, it is difficult to ascertain the role of Brd2 in segregation based on their yeast data. Mutations in MeCP2 have been linked to cause the severe neurodevelopmental Rett syndrome (RTT) (Amir et ah, 1999). MeCP2 binds directly to symmetrically methylated CpG dinucleotides via a conserved methyl binding domain (MBD) (Free et al, 2001; Lewis et ah, 1992; Nan et ah, 1997). It also contains a transcriptional repression domain (TRD), shown to bind proteins such as histone deacetylases (HDACs), which serves to inhibit transcription in a subset of genes (Shabazian and Zoghbi, 2002; Tudor et ah, 2002; Ballestar et ah, 2005). The C- terminal domain (CTD) of MeCP2 is required for mediating chromatin-specific interactions, binding histones, and compacting chromatin (Nikitina et ah, 2007 and references therein). In vitro studies indicate that MeCP2 is an intrinsically disordered protein where even the secondary structure of MBD and TRD are 40% and 85% unstructured, respectively (Adams et ah, 2007). Nonetheless, MBD, TRD and CTD have been shown to bind non-specifically to both methylated and unmethylated DNA (Adams et ah, 2007; Nikitina et ah, 2007). It is thought that the ability of MeCP2 to simultaneously contact multiple DNA sites as well as histones explains the formation of secondary and tertiary chromatin structures upon incubating MeCP2 with nucleosome-bound DNA (Adams et ah, 2007; Nikitina et ah, 2007). The modularity of MeCP2 and its ability to bind non-specifically to DNA suggests that it could bind randomly to plasmids and concurrently tether them to mitotic chromosomes, thereby governing their stable partitioning in yeast cells. This mode of plasmid maintenance would not depend on EBNA1 or the FR element, consistent with the results observed in the yeast plasmid loss assay (Figure 11). 83 Silencing Brd2 and MeCP2 did not change EBNA1 localization on DNA during the cell- cycle (Figure 19), suggesting that Brd2 and MeCP2 are not involved in tethering EBNA1 to chromatin. It should be noted that the silencing of MeCP2 was poor so it would be worth investigating other more efficient techniques to down-regulate this protein before further conclusions regarding its role in mediating EBNA1 attachment to DNA can be drawn. Nevertheless, given that Brd2 and MeCP2 are able to maintain plasmids independently of EBNA1, this implies that the proteins themselves might be able to tether plasmids to cellular DNA. For this reason, it would be important to perform biochemical fractionation experiments followed by Southern blot analyses to determine whether or/P-containing plasmids are released from the chromosomal pellet upon silencing Brd2 and MeCP2. It is also worth mentioning that while plasmid attachment to mitotic chromosomes represents one mechanism to explain plasmid stability observed in yeast cells, it is not the sole mechanism. It is possible that these proteins increase plasmid replication, which would result in high-copy numbers and increased plasmid retention in cells. However, since neither protein has been documented to play a role in replication, this explanation might not be likely. IV.3. The Role of Brd4 in EBNA1-Mediated Segregation in Budding Yeast Brd4 is a large, multi-functional protein with roles in cell-cycle progression, transcription and DNA replication (reviewed in Wu and Chiang, 2007). Recently, Brd4 has become a subject of great interest because it has been proposed to be a key player in viral persistence. First, it has been implicated in KSHV segregation since it interacts with LANA1 and the two proteins co- localize as discrete punctate spots on cellular mitotic chromosomes (You et at, 2006; Ottinger et at, 2006). Second, using a similar yeast plasmid loss assay described in my thesis, Brd4 has been shown to reconstitute BPV1 E2-mediated plasmid maintenance (Brannon et at, 2005). Third, it has been implicated in segregation of other human PVs (although not all) based on the ability of the E2 proteins to co-localize with Brd4 on host mitotic DNA (Oliveira et at, 2006; McPhillips et at, 2006). These observations coupled to the fact that Brd4 belongs to a rare group of transcription factors that remain associated with DNA during mitosis (Dey et at, 2000; 2003), make Brd4 a likely candidate for mediating EBNA1-dependent segregation of EBV genomes. In my studies, I successfully showed that mBrd4 can reconstitute YRp7FR maintenance in actively-dividing yeast cells provided that EBNA1 is also present (Figure 13). 84 Thus, it seems like multiple viruses are able to utilize Brd4 in order to mediate stable plasmid partitioning. Given that EBNA1 and Brd4 are able to interact (Figure 16), it is likely that the mechanism by which segregation occurs is through Brd4-mediated tethering of EBNA1 and YRp7FR plasmid to mitotic chromosomes. In yeast, there are two homologues of Brd4, yeast bromodomain factor 1 and 2 (Bdflp and Bdf2p), both of which are short members of the BET family of proteins. These proteins have a predicted molecular mass of-77 kDa and -72 kDa, respectively, and have been shown to run at -100 kDa in SDS-polyacrylamide gels (Matangkasombut et ah, 2000). Thus, they are not likely to be the non-specific protein band that runs at -130 kDa observed in the anti-Brd4 Western blot (Figure 13B). Bdfl mutant cells are viable as long as Bdf2p is present, but cells cannot survive if both Bdflp and Bdf2p are absent (Matangkasombut et ah, 2000), suggesting that the two proteins may have redundant roles. However, bd/2 mutants exhibit no defects while bdfl mutants have defects in snRNA transcription, vegetative growth, meiosis and also exhibit a temperature-sensitive lethality (Lygerou et ah, 1994; Chua and Roeder, 1995). This suggests that Bdf2p is redundant for some Bdflp functions, but not all (Chua and Roeder, 1995). Moreover, while both Bdflp and Bdf2p have roles in transcription and are able to interact with mitotic chromosomes (Lygerou et ah, 1994; Matangkasombut et ah, 2000), each has different preferences for acetylated histone tails (Ladurner et ah, 2003; Matangkasombut and Buratowski, 2003). As BET family members, both Bdflp and Bdf2p contain two bromodomains and an ET domain. A sequence alignment comparing the bromodomains and ET domains of Bdflp and Bdf2p with that of hBrd4 shows that there is -20-40% protein sequence identity (data not shown; see Figure 1 of the Supplementary material in Wu and Chiang, 2007). Since YRp7FR cannot be maintained in the presence of EBNA1 alone, this suggests that neither Bdflp nor Bdf2p can mediate plasmid partitioning in yeast. The inability of Bdflp and Bdf2p to interact with EBNA1 is a likely explanation for the observed negative segregation activity. Indeed, in the BPV1 system, E2 interacts with the C-terminal domain of Brd4 (You et ah, 2004) not present in Bdflp and Bdflp cannot maintain BPV1-based plasmids even in the presence of E2 (Brannon et ah, 2005). However, a fusion protein of Bdflp containing the C- terminal tail of Brd4 was able to partially rescue plasmid stability (Brannon et ah, 2005), presumably because the interaction has been restored. Similarly, one could test whether the lack of EBNA1 binding to Bdflp is the reason that this protein does not support segregation by 85 mapping the EBNA1-binding domain of Brd4, then creating a fusion protein between Bdflp and the EBNA1-binding domain of Brd4 and testing if this could rescue plasmid maintenance in the presence of EBNA1. In order to gain insight into the mechanism by which mBrd4-mediated segregation occurs in yeast, I attempted to map the region of EBN A1 that is required for this function. The results from the yeast plasmid loss assay indicate that while deletion of residues 8-67 or 61-83 abolish YRp7FR plasmid partitioning in yeast, deletion of residues 325-376 showed slightly reduced plasmid maintenance effects compared to wild-type EBNA1 (see Table 1 for a summary of the plasmid loss assay results). Thus, the N-terminal region of EBNA1 spanning amino acids 8-83 is required for mBrd4-mediated segregation while the central Gly-Arg region spanning amino acids 325-376 contributes to this activity. These results were surprising since amino acids 61-83 were previously established as a domain required for transactivation and amino acids 325-376 were shown to be required for segregation in human cells (Wu et ah, 2002; Kapoor et ah, 2001). Nevertheless, these observations suggest that in human cells, Brd4 plays a smaller role in modulating episome segregation since EBNA1A61-83 has wild-type segregation activity (Wu et ah, 2002; also see Discussion Section IV.4.). Moreover, these results lead me to speculate that Brd4 might be involved in EBNA1-mediated transactivation in human cells (see Discussion Section IV.5.). To determine if the failure of the EBNA1 mutants to mediate plasmid partitioning was due to a failure of the EBNA1 mutants to interact with Brd4, I conducted co-IP experiments to map the region of EBNA1 involved in Brd4 binding (see Table 1 for a summary of the co-IP results). The results confirmed that EBNA1 and EBNA1A325-376 were able to bind Brd4 although the interaction was weak. Furthermore, the inability of EBNA1A61-83 to confer plasmid stability was consistent with the lack of interaction with Brd4. However, I was surprised to discover that EBNA1A8-67 and the double deletion mutant EBNA1A8-67/325-376 both interacted more strongly with Brd4 than wild-type EBNA1, even though EBNA1A8-67 cannot support mBrd4-mediated segregation in yeast. These results indicate that residues 8-67 are required for segregation but not for the interaction between EBNA1 and Brd4. Taken together, these observations shed light into the mechanism by which Brd4 mediates plasmid stability in yeast cells. First, the results map the minimal Brd4-binding region of EBNA1 to residues 67-83. Since deletion of amino acids 8-67 increases binding to Brd4, it is 86 Table 2. A summary of the effects of EBNA1 and EBNA1 mutants on mBrd4-mediated segregation of the FR plasmid in yeast and their interactions with Brd4 in human cells. Yeast Plasmid Loss Assay Interaction with Brd4 EBNA1 and Mutants (% Colonies Maintaining (Apparent strength of Plasmids ± SD)a interaction) EBNA1 Yes (63 ± 21) Yes (weak) EBNA1A325-376 Yes (49 ± 20) Yes (weak) EBNA1A8-67 No (4 ± 4) Yes (strong) EBNA1A8-67/325-376 N/A Yes (strong) EBNA1A61-83 No (6 ± 4) No 452-641 N/A No aSee Figures 13 and 14 for plasmid loss assay results. See Figure 16 for Western blots from co-immunoprecipitation experiments using 293T cells. 87 likely that this deletion exposes this adjacent site, thereby increasing its availability for the interaction between EBNA1 with Brd4. Second, my results also suggest that a weak or transient interaction between Brd4 and EBNA1 (i.e. wild-type EBNA1 and EBNA1A325-376) allows for optimal segregation activity. Conversely, a strong interaction between Brd4 and EBNA1 (specifically, EBNA1A8-67) may be detrimental for plasmid maintenance. Thus, Brd4-mediated plasmid segregation might require a transient association between Brd4 and EBNA1 at a particular time in the cell cycle. Indeed, having just the right strength of interaction could ensure that EBNA1 is transiently tethered to chromosomes during mitosis and then efficiently released for the next round of plasmid replication. Such a transient interaction might explain why I have not been able to detect an in vivo association between endogenous EBNA1 and Brd4 in EBV-positive Burkitt's lymphoma RAJI cells or EBV-positive nasopharyngeal carcinoma C666-1 cells. Furthermore, there is precedent to support the contention that the association between two proteins must be tightly regulated in order achieve optimal episome segregation. Voitenleitner and Botchan (2002) showed that for BPV1, proper E1-E2 complex formation is a prerequisite for E2 localization on mitotic chromosomes and stable partitioning of BVP1-based plasmids in cells. El does not co- localize with E2 on host mitotic chromosomes although an association between the two proteins is required during interphase for episome replication (Voitenleitner and Botchan, 2002, and references therein). The authors show that an E2 mutant that binds stronger to El compared to wild-type E2 is defective in binding mitotic chromosomes, which results in defective plasmid segregation. Therefore, the strength of the E1-E2 interaction must be regulated to ensure plasmid maintenance. Similarly, I propose that the EBNA1-Brd4 association must also be properly regulated to ensure that EBNA1 is able to carry out its roles after mitosis. In addition to the regulation of the strength of interaction between Brd4 and EBNA1, the total amount of EBNA1 protein present in cells might also contribute to segregation. Although results are still preliminary, it was interesting that low EBNA1 levels in yeast supported segregation to a greater extent than high EBNA1 levels (see Figure 14B and Results Section III.5.). This effect could be explained by the fact that high levels of EBNA1 might be toxic to cells. Alternatively, in keeping with the idea that a weak association between Brd4 and EBNA1 allows for optimal segregation activity, it is possible that low EBNA1 levels in yeast ensure that the interaction between EBNA1 and Brd4 is kept transient. Therefore, plasmid stability is 88 maintained in yeast cultures expressing the lowest amount of EBNA1 protein. Consistent with this, our laboratory has found that human cell lines stably expressing low levels of EBNA1 are able to maintain or/P-containing plasmids more efficiently than cell lines expressing high levels of EBNA1 from an exogenous plasmid (Sivachandran and Frappier, unpublished data). Nevertheless, this hypothesis will still require further investigation. The results from the yeast plasmid loss assay and the co-IP experiments provide a third insight into the regulation of Brd4 and EBNA1-mediated segregation in yeast. Given that EBNA1 residues 8-67 and 61-83 are required for plasmid partitioning, yet only residues 61-83 are required for the interaction between EBNA1 and Brd4, the mechanism by which each region of EBNA1 functions in segregation must differ. Indeed, 61-83 has a more direct role in segregation since it encompasses the minimal Brd4-interaction domain of EBNA1. This interaction is likely required for mitotic chromosome association and subsequent plasmid partitioning. However, 8-67 likely has an indirect effect on segregation as it regulates the weak or transient interaction between EBNA1 and Brd4. The presence of residues 8-67 probably masks the minimal Brd4-interaction domain of EBNA1 and hinders the association between the two proteins. This regulation would allow for the timely association and dissociation between EBNA1 and Brd4, which could allow for optimal segregation activity. In summary, my studies in yeast have provided further insight into the DNA segregation mechanism of EBV. It is now clear that EBNA1 is able to use multiple protein partners (i.e. hEBP2 and Brd4) in order to achieve stable episome persistence. Interestingly, the domains of EBNA1 that are required for hEBP2-mediated segregation differ from those of Brd4 (see Figure 20), further providing evidence that EBNA1 can utilize multiple mechanisms to govern efficient plasmid partitioning. My studies also support that the role of Brd4 in segregation of viral episomes could be evolutionarily conserved among small double-stranded DNA viruses. As such, investigating the mechanisms by which viral proteins commandeer Brd4 could prove useful in the development of anti-viral drugs and gene therapy techniques. IV.4. The Role of Brd4 in Segregation in Human Cells Using yeast as a model system for studying EBV segregation has the advantage that the isolated system allows individual cellular proteins to be tested for their effects on plasmid 89 DNA-binding and 18 33 53 67 101 325 376 386 459 607 641 EBNA1 Domains Involved in Mediators of Segregation Segregation 8 67 325 376 459 607 8 83 325 376 459 607 I | Contributes H Required Figure 20. Different domains of EBNA1 are required for hEBP2 versus mBrd4-mediated segregation of the FR plasmid in yeast. The structural domains of EBNA1 are indicated. Black boxes indicate regions of EBNA1 that are required for segregation in yeast cells and grey boxes indicate regions of EBNA1 that contribute to segregation. Both modes of segregation required the C-terminal DNA-binding and dimerization domain of EBNA1. hEBP2-mediated segregation requires EBNA1 amino acids 325-376 and residues 8-67 contribute to this activity. Conversely, mBrd4-mediated segregation requires EBNA1 amino acids 8-83 and residues 325- 376 contribute to this activity. 90 stability. However, the disadvantage is that an isolated system may not fully represent the events that occur in complex mammalian cells. In other words, while EBNA1 is able to utilize multiple, independent mechanisms in order to achieve segregation in yeast, it is unclear if all mechanisms are important in human cells. The fact that hEBP2 binds EBNAl through amino acids 325-376 (Kapoor et al, 2001) and that these residues have been shown to be required for plasmid maintenance and EBNAl association with mitotic chromosomes in human cells (Wu et al, 2000; 2002), suggest that hEBP2 might have the greater role in EBV segregation compared to Brd4. Nonetheless, it is still important to assess the role of Brd4 in segregation in human cells since it is still possible that Brd4 works in conjunction with hEBP2. To this end, I asked if Brd4 could play a role in EBNAl attachment to mitotic chromosomes. The results showed that silencing Brd4 had no effect on EBNAl association with cellular DNA (Figure 17 and 18); this contrasted with previous results where silencing hEBP2 caused EBNAl to dissociate from mitotic chromosomes (Kapoor et al, 2005). Despite these observations, four explanations can be provided in support of the possibility that Brd4 can still play a role in EBV segregation. First, while Brd4 silencing can be detected by Western blot, it is possible that the down-regulation was not efficient enough to see an effect on EBNAl attachment to mitotic chromosomes. Others have shown that injecting anti- Brd4 antibodies blocked cells in the G2-M phase (Dey et al, 2000; 2003; Muruyama et al, 2002). I did not observe this phenotype upon treating cells with esiBRD4, likely due to insufficient Brd4 down-regulation. Thus, the residual Brd4 was likely sufficient to mediate EBNAl tethering. Second, it is likely that the role of Brd4 in EBV segregation is redundant and/or minor compared to other mediators of episome partitioning. As suggested earlier, the yeast system represents an isolated system where one mediator protein is tested at a time with EBNAl. However, in the human system, a whole array of potential mediator proteins is present. EBNAl may utilize certain proteins in preference over others when given a choice. Perhaps using Brd4 is not the preferred choice; thus, silencing Brd4 did not show any effect on EBNAl association with mitotic chromosomes. The fact that Brd4 interacts with residues 61-83 of EBNAl, a region not required for mitotic chromosome association or segregation function (Wu et al, 2002), provides the first clue in support of this hypothesis. Morever, metaphase spreads performed in our laboratory using the EBV-positive C666-1 cell line revealed little co-localization between 91 EBNA1 and Brd4 on mitotic chromosomes (Shire and Frappier, unpublished data). This provides additional evidence that Brd4 does not have a large role in EBNA1 attachment to DNA, consistent with my silencing and fractionation data. Alternatively, if there are multiple parallel pathways to ensure EBNA1 association with mitotic chromosomes, it is likely that eliminating one pathway (i.e. the Brd4 pathway) is not sufficient to abolish EBV segregation. Third, while Brd4 does not appear to be involved in EBNA1 attachment to DNA, it could still be involved in episome partitioning. The biochemical fractionation experiments conducted in my studies do not test plasmid partitioning directly. In order to thoroughly assess the role of Brd4 in episome segregation in human cells, it would be important to silence Brd4 long enough to allow for the EBNA1-containing cells to undergo several rounds of cell division. Then, these cells can be harvested and the amount of on'P-plasmids harboured by these cells can be quantified and compared to control cells. Fourth, while EBNA1 remains associated with DNA when Brd4 is silenced, it is not certain if EBNA1 is still functional for segregation. That is, without assessing plasmid localization by biochemical fractionation, it is not known if the plasmid remains associated with mitotic DNA when Brd4 is silenced or if it dissociates because EBNA1 function is altered in the absence of Brd4. This latter scenario would imply that an EBNA1-Brd4 association is not required for EBNA1 attachment to mitotic chromosomes, as other mediator proteins can probably perform this role in the absence of Brd4. However, the association might be required for a functional EBNA1 to tether viral episomes to mitotic DNA. Therefore, at this point, it cannot be concluded that Brd4 does not play any role in EBV segregation in the human system. Nonetheless, it is still necessary to address the possibility that Brd4 does not play a role in segregation in the human system despite the observations from the yeast system. The yeast plasmid loss assays were conducted using the mouse homologue of Brd4. Although mouse and human Brd4 share 93% protein sequence identity (see Figure 12), they could still play different roles in EBNA1-mediated segregation. Alternatively, perhaps the yeast data is an artefact of over-expressing mBrd4. To discount these possibilities, it would be necessary to integrate both EBNA1 and human Brd4 into yeast and monitor the stability of YRp7FR under conditions of lower EBNA1 and Brd4 levels. Of interest, during the course of my studies, I was able to show that mBrd4 can reconstitute plasmid maintenance in a yeast strain with integrated EBNA1 (data not shown). Lastly, Brd4 may indeed play a role in segregation in the yeast system. However, 92 the Brd4-EBNA1 interaction may serve other purposes in human cells (discussed in the next section). IV.5. Brd4 May Have Multiple Roles in the EBV Life Cycle The EBNA1 mutant, EBNA1A61-83, has full segregation activity in human cells but is defective in transcriptional activation (Wu et al, 2002). The finding that Brd4 interacts with EBNA1 through this region suggests that Brd4 could have a role in EBNA1-mediated transcription activation. In support of this idea, our laboratory has shown that silencing or over- expressing Brd4 in human cells expressing EBNA1 resulted in a decreased ability of EBNA1 to activate the expression of a CAT reporter gene under control of the EBV FR element (Wang and Frappier, unpublished data). This suggests that Brd4 is a positive regulator of EBNA1 -mediated transcription. Morever, ChIP experiments showed that Brd4 localized to the FR element but not the DS element of oriP in asynchronously-growing EBV-positive Raji cells (Wang and Frappier, unpublished data). This is expected if Brd4 is involved in EBNA1-mediated transactivation. These results are exciting since Brd4 is the first cellular protein known to bind EBNA1 through residues 6.1-83. Furthermore, the finding that Brd4 is involved in EBNA1-mediated transactivation suggests that Brd4 might be the missing co-factor with which EBNA1 governs transcription in human cells. It is also important to point out that the ChIP data correlates with the data from the yeast plasmid loss assay, as finding Brd4 associating with oriP raises the possibility that EBNA1 and Brd4 may together bind the FR element to mediate stable episome segregation. Taken together, these results strongly suggest that Brd4 may have multiple roles in the EBV life cycle, which include transcription and segregation. It has been proposed that Brd4 may also play multiple roles in the life cycle of BPV1 and KSHV. It has been shown that Brd4 interacts with the transcription activation domain of E2 and that this interaction is required for E2-mediated transcription activation (McPhillips et al, 2006; Schweiger et al, 2006; lives et al, 2006). Furthermore, similar to the case for EBV, it seems that the E2-Brd4 association is more important for transcription than segregation since abrogating the association does not disrupt E2 attachment to mitotic chromosomes for all papillomaviruses, but it does abolish transcription activity for all papillomaviruses (McPhillips et al, 2006). For KSHV, Brd4 has been implicated to play a role in LANA1-mediated segregation (You et al, 2006; also see Introduction Section 1.3.2.). Moreover, others have shown that 93 LANA1 can transactivate a luciferase reporter gene containing a CDK2 promoter (Wong et ah, 2004), but this activity is inhibited in the presence of Brd4 in a dose-dependent manner (You et ah, 2006). Thus, not only is Brd4 a common target for multiple viruses, it also appears that Brd4 has a complex role in the life-cycle of these viruses. The interaction between Brd4 and the viral protein of interest is therefore a potential target for the development of antiviral therapeutics. IV.6. Assessing the Ability of EBNA1 to Bind DNA Throughout the Cell Cycle The biochemical fractionation experiments presented in this study revealed that EBNA1 associates with the chromatin pellet throughout the cell cycle (Figures 17 and 18). While EBNA1 loading onto chromatin during G2 and its association with condensed chromosomes during mitosis are required for EBV genome persistence, the importance of EBNA1 binding to chromatin during interphase is unclear. It is possible that placing EBNA1 on chromatin at this stage of the cell-cycle ensures that the viral genome gains access to the host replication machinery and it is therefore able to be efficiently amplified. Indeed, this has been proposed for BPV1 E2, which is found on chromatin throughout the cell cycle (lives et ah, 1999). However, an EBNA1 mutant (EBNA1A325-376) that is defective in chromosome binding shows wild-type replication activity (Wu et ah, 2002). This suggests that chromosome binding is not a pre requisite to EBNA1-mediated replication. Alternatively, it also suggests that chromatin- association during interphase might occur through a different domain of EBNA1. For this reason, I asked if Brd4, which interacts with EBNA1 through residues 61-83, could be involved in tethering EBNA1 to interphase chromatin. To this end, I fractionated asynchronously-growing HeLa cells that were treated with esiBRD4. The results indicated that Brd4 has no such role as EBNA1 remained associated with chromatin upon Brd4 down-regulation (Figure 18). It has also been proposed that EBNA1 association with chromatin during Gl-S places the viral genome at a location which will ensure its proper partitioning during mitosis (Kanda et ah, 2001; Kanda et ah, 2007; Nanbo et ah, 2007). Thus, whether EBNA1 association with interphase chromatin is important for replication or segregation or both will require further investigation. It is important to mention that it is not clear if EBNA1 localization in the pellet fraction of the biochemical fractionation experiment is due to chromatin binding or pelleting for other reasons such as being insoluble under the low salt conditions used (15 mM KC1). This issue could be addressed by treating nuclear pellets with DNasel and analyzing the soluble proteins for 94 the presence of EBNA1. If EBNA1 is indeed chromatin-bound, it will be found in this DNasel- treated soluble fraction. Conversely, if EBNA1 precipitates as a result of being insoluble, then even after DNasel digestion, EBNA1 will still be found in the pellet fraction. These studies are currently on-going in our laboratory. It is worth mentioning that Kanda and colleagues performed a similar biochemical fractionation experiment on log-phase cells using 10 mM KC1 and they successfully released EBNA1 from the chromatin pellet fraction upon treatment of nuclei with micrococcal nuclease (Kanda et al, 2001). Thus, pelleting of EBNA1 under these low salt conditions appeared to be due to association with chromatin. IV.7. Multiple Pathways and Mechanisms to Support EBV Episome Partitioning Recently, a former graduate student in our laboratory showed using immunofluorescence microscopy that the co-localization of EBNA1 and hEBP2 on mitotic chromosomes occurred in the later half of mitosis from metaphase to telophase (Nayyar and Frappier, unpublished data). EBNA1 associated with condensed cellular DNA as early as prophase but hEBP2 was still in the process of diffusing out of the nucleolus at this time. This discovery provided further support for the existence of other mediator proteins that tether EBNA1 to mitotic chromosomes. Moreover, one (or more) of these potential mediators would act early in the stages of mitosis from prophase to metaphase. The ATP-dependent DNA helicase, ChlRl, was shown to play a crucial role in "loading" BPV1 E2 onto host mitotic chromosomes early in mitosis (Parish et al, 2006; see Introduction Section 1.3.1.). Given that viruses are able to utilize common cellular targets to perform their functions, it would be worthwhile to investigate if ChlRl also co-localizes with EBNA1 early in mitosis and if this association is important for EBV segregation. Nonetheless, these results suggest that the mitotic tethering model might include a possible two-step process that involves early-phase mitotic tethering and late-phase mitotic tethering. In this case, hEBP2 would be considered as a mediator of late-phase mitotic tethering. It is important to note that some mediator proteins may serve to tether EBNA1 to mitotic chromosomes throughout all stages of mitosis. However, other mediator proteins may serve to load EBNA1 onto DNA early in mitosis, after which other additional interactions are required to maintain this association. My investigation into the DNA segregation mechanism of EBV has uncovered novel mechanisms that might be involved in EBV segregation. Figure 21 illustrates a schematic of the possible mechanisms. It is known that hEBP2 plays an important role in tethering EBNA1 and 95 EBV-based plasmids to mitotic chromosomes. My studies have shown that Brd4 may also have a role in EBV segregation although its role compared to hEBP2 is likely minor in human cells. Given that EBNA1 and Brd4 are able to interact and that Brd4 binds chromosomes, the mitotic tethering model might also be used to explain Brd4-mediated segregation. My studies also show that Brd2 and MeCP2 may have more general roles in plasmid partitioning that is EBNA1 and FR-independent. Both proteins might interact with the plasmid and tether it to mitotic chromosomes in order to ensure its stable partitioning. It is also possible that the Gly-Arg rich regions of EBNA1 could contribute to chromatin interactions by contacting DNA directly even though data indicate that this could not be sufficient for mitotic chromosome attachment. Nevertheless, it is possible that direct DNA interactions function to place EBNA1 on chromosomes prior to mitosis and then other cellular proteins serve to stabilize this association as the cell progresses through mitosis. In conclusion, my studies indicate that EBV has likely evolved multiple mechanisms that work in parallel or in conjunction with one another to ensure persistent infection. 96 A 11 / i Figure 21. Multiple proposed mechanisms of EBV segregation. The results from these studies indicate that EBV likely utilizes multiple mechanisms in order to ensure faithful partitioning of its EBV genome to daughter cells during cell division. Previously, hEBP2 has been shown to mediate attachment of EBNA1 to mitotic chromosomes. Brd4 may also perform the same role although probably minor compared to hEBP2. Brd2 and MeCP2, both of which are general mediators of plasmid segregation in yeast, may tether the EBV genome to host mitotic chromosomes in a non-specific fashion. 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