UNIVERSITY OF CINCINNATI February 6 , 2002 I, Stacey K. Ogden, hereby submit this as part of the requirements for the degree of: Doctor of Philosophy in: Molecular Genetics, Biochemistry and Microbiology It is entitled: HBx-mediated disruption of p53 tumor suppressor function leading to reactivation of a silenced tumor- marker gene. Approved by: Dr. Michelle Barton, Chair Dr. Tony Capobianco Dr. Iain Cartwright Dr. Michael Lieberman Dr. Jun Ma HBx-mediated disruption of p53 tumor suppressor protein function leading to re-activation of a silenced tumor marker gene A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph. D.) in the Department of Molecular Genetics, Biochemistry and Microbiology of the College of Medicine 2002 by Stacey K. Ogden B.A., Miami University, 1997 Committee Chair: Michelle Craig Barton, Ph. D. Abstract Chronic infection with Hepatitis B Virus (HBV) is a predominant risk factor associated with development of hepatocellular carcinoma (HCC). Individuals who are chronically infected with HBV are over 200-times more likely to develop HCC than those who are not infected. Multiple studies have implicated the virally encoded X protein (HBx) as the candidate oncoprotein responsible for cellular transformation. HBx forms a complex with cellular p53 tumor suppressor pr otein to result in modification of p53-mediated gene regulation, DNA damage detection and modulation of apoptosis. The developmentally silenced a-fetoprotein (AFP) tumor marker gene, which is transcriptionally repressed by p53, is tightly correlated with HCC development: it is reactivated in over 80% of all liver carcinomas. p53 mediates transcriptional repression of AFP through an over- lapping HNF-3/Smad4/p53 binding element located within the developmental repressor domain of the AFP promoter. Here, us ing AFP as a model gene, we have examined the mechanism by which p53 facilitates transcriptional repression, and how this repression is disrupted upon p53-HBx interaction. In vitro chromatin assembled transcription analysis and enzyme accessibility studie s demonstrate that p53 association at the overlapping binding element is required during chromatin assembly for reorganization of AFP promoter chromatin structure to result in occlusion of restriction enzymes and general transcription factors from the transcription start site. Protein-DNA binding assays show that p53 association at this element is required to recruit mSin3A co-repressor and stabilize association of a putative Smad4 and SnoN containing co-repressor complex with AFP chromatin templates. HBx-mediated reactivation of AFP is achieved through direct p53-HBx interaction resulting in disruption of SnoN co-repressor binding to AFP chromatin templates. Table of contents Chapter 1: Introduction ..................................................................................................................3 Chapter 2: p53 targets chromatin structure alteration to repress α-fetoprotein gene expression.19 Figure 1. p53 represses AFP chromatin transcription in vitro.............................................32 Figure 2. p53 organizes chromatin structure.........................................................................33 Figure 3. DNA binding is required for p53 function. ..........................................................35 Figure 4. p53 mediated repression of AFP transcription occurs even in the presence of hyperacetylated histones at the core promoter......................................................................37 Chapter 3: Hepatitis B viral transactivator HBx alleviates p53-mediated repression of α- fetoprotein gene expression .........................................................................................................39 Figure 1: HBx and p53 interaction results in loss of p53-mediated repression of AFP transcription. .........................................................................................................................62 Figure 2: p53-mediated squelching of β-globin transcription is not alleviated by HBx.......63 Figure 3: HBx alleviates p53 mediated repression of chromatin assembled AFP DNA. .....64 Figure 4: p53 repression and HBx re-activation of AFP transcription are dependent upon p53-DNA binding. ................................................................................................................66 Figure 5: Tissue specificity of p53-HBx effect on AFP transcription is maintained in chromatin. .............................................................................................................................68 Figure 6: HBx associates with DNA-bound p53. (A) p53-DNA binding is maintained upon HBx association....................................................................................................................70 Chapter 4: A p53, Smad4 and SnoN-containing repressor complex is disrupted by the virally encoded HBx protein. ..................................................................................................................72 1 Figure 1: SnoN associates at the AFP p53 regulatory element.............................................97 Figure 2: Putative co-repressor complex associates at the p53 regulatory element............100 Figure 3: Smad4 and SnoN association with p53 are DNA-dependent..............................101 Figure 4: SnoN is involved in AFP transcription repression..............................................103 Figure 5: p53 DNA binding is required for maximal SnoN-mediated AFP repression......105 Figure 6: Functional p53 and SnoN are both required for maximal AFP repression. ........107 Figure 7: HBx disrupts p53-stabilized SnoN binding to chromatin DNA templates. ........109 Chapter 5: Summary and Conclusions.......................................................................................110 Figure Summary..................................................................................................................115 References..................................................................................................................................116 2 Chapter 1: Introduction p53 and Cancer It has been suggested that in order for a cell to be considered to have a cancer phenotype, it must possess six distinctive traits: self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replication potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan and Weinberg, 2000). The processes by which a cell acquires these traits are complex and, at present, not clearly understood. However, it is clear from multiple lines of evidence that loss of genomic integrity within tumor cells can contribute to development of these cancer traits. Safeguarding genomic integrity is normally controlled by a host of cellular proteins; the most prominent being the p53 tumor suppressor protein. Functional p53 protein has a vast array of roles involved in detection and repair of DNA damage, cell cycle arrest and modulation of apoptosis (reviewed in Ko and Prives, 1996). The importance of p53 in controlling proper cellular growth is directly evidenced by the fact that it is the single most commonly mutated or deleted gene in human cancer. p53 exhibits a mutation rate well over 80% in human tumors (reviewed in Fisher, 2001; Ko and Prives, 1996). Tumor cells with inactive p53 protein typically exhibit increased aneuploidy, DNA mutation and gene amplification in combination with decreased apoptotic potential (reviewed in Kastan, et al., 1995). Central to p53’s ability to regulate cell cycle control and safeguard against mutation is its ability to activate and repress appropriate target genes upon detection of DNA damage. Among the genes activated by p53 protein in response to cellular stress are p21, mdm-2, GADD45, cyclin G, bax, and IGF-BP3. The ability of p53 to regulate its target genes requires direct DNA binding. This function is lost in the bulk of p53-null tumors. Most p53 loss-of-function 3 mutations occur within the DNA binding domain (reviewed in Ko and Prives, 1996). Because most of the above-mentioned p53 regulated genes are either directly or indirectly involved in DNA damage repair and/or cell cycle arrest, loss of p53 DNA binding and transcription control affords a growth advantage to tumor cells. In fact, p53 DNA binding mutants can act as “dominant negative” factors in some cell lines. DNA binding mutants form oligomeric complexes with wild type p53 protein and/or transcription co-activators to prevent their binding and activating target genes. This is believed to be one mechanism by which mutant p53 protein transforms cultured cells in tumorigenesis assays (reviewed in Levine, et al., 1991). In addition to activation of target genes, p53 is also involved in both direct and indirect transcription repression. Multiple viral genes and a handful of cellular genes have been demonstrated to be indirectly repressed by p53 protein. Among the cellular genes indirectly repressed by p53 are c-fos, c-jun, IL-6, Rb and Bcl-2. These genes lack direct p53-DNA binding sites, and are likely repressed by over-expressed p53 through its ability to sequester general transcription factors and co-activators of transcription. This “broad-range” ability of p53 to regulate transcription repression is likely important to p53 tumor suppressor function, as these cellular gene products