UNIVERSITY OF CALIFORNIA, SAN DIEGO Biological Function of the LxCxE-binding Pocket of Retinoblastoma Protein A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Jacqueline Bergseid Committee in charge: Professor Jean Y. Wang, Chair Professor Randall S. Johnson, Co-Chair Professor Ju Chen Professor Cornelis Murre Professor Geoffrey Wahl 2007 UMI Number: 3244732 UMI Microform 3244732 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 Copyright Jacqueline Bergseid, 2007 All rights reserved The dissertation of Jacqueline Bergseid is approved, and it is acceptable in quality and form for publication on microfilm: Co-Chair ________________________________________________________________________ Chair University of California, San Diego 2007 iii DEDICATION To the people who made this work possible: my mother, who taught me the value of persistence, and my husband, whose love carried me through. iv TABLE OF CONTENTS Signature Page.…iv Table of Contents…………………………………………………………………….……v List of Figures……………………………………………………….………vi List of Tables………………………………………………………….ix Vita and Publications………………………………………………………………….xii Chapter I: Introduction………………………………………………………….…………1 Chapter II: Generation of RbN750F mice………………………………………………….17 Chapter III: Phenotype of RbN750F/N750F MEFs and 3T3s……………………………….40 Chapter IV: Phenotype of Rb+/N750F, RbN750F/N750F and RbN750F/- mice………………….65 Chapter V: General discussion………………………………………………….113 v LIST OF FIGURES Page Chapter I Figure 1.1 Structure of RB protein……………………………………….2 RB protein regulates entry into S phase of the cell cycle …………….3 Two models of RB function………………….10 Chapter II Figure 2.1 Cloning strategy for generation of the targeting construct…………….2 Addition of the 3’ extension using recombineering……………….3 Insertion of the Neo cassette using recombineering…………………….4 Generation of Rb+/N750F ES cells…………………………………….5 Testing for the germ line transmission and breeding of RbN750F/N750F mice………………………………………………………………………36 Figure 2.6 Genotyping PCR for transmission of RbN750F allele and Neo cassette excision………………………………………………………….………37 Chapter III Figure 3.1 pRb-N750F does not bind to Adenovirus E1A……………………….2 Terminally differentiated RbN750F/N750F myotubes are resistant to re- stimulation with serum…………………………………….3 Accelerated immortalization of RbN750F/N750F MEFs………………….4 RbN750F/N750F MEFs exhibit normal cell cycle progression and respoind to contact inhibition…………………………………….5 RbN750F/N750F MEFs do not exhibit upregulation of cell cycle genes whose expression is increased by infection with Adenovirus E1A protein…….61 Chapter IV Figure 4.1 Histological analysis of the skeletal and cardiac muscle…………….2 Platelet counts in the peripheral blood………………………………….3 Differentiation of hematopoietic stem cells………………….4 Analysis of progenitor cells in the platelet linage…………………….5 Lymphocyte counts in the peripheral blood…………………………….6 Relative ration of T lymphocytes to B lymphocytes in the peripheral blood of RbN750F/N750F mice……………………………………………….7 Histological analysis of the spleen…………………………………….8 Histological analysis of the ovaries from RbN750F/N750F females ….9 RbN750F/- mice show a characteristic hunchback posture…………….10 Early mortality and reduced mass of RbN750F/- mice……………….11 Histological analysis of the ovaries from RbN750F/- females…………….12 Histological analysis of the testis from RbN750F/- males………….107 vii LIST OF TABLES Page Chapter II Table 2.1 Results of blastocysts injections………………………………….2 Mendelian distribution of RbN750F allele ……………………………….29 Chapter III Table 3.1 Deregulation of pattern-formation genes in RbN750F/N750F MEFs …….51 Chapter IV Table 4.1 Mendelian distribution of RbN750F allele ……………………………….2 Tumor-free survival of Rb+/N750F and RbN750F/N750F mice ……………….3 Complete blood cell count (129/B6 mixed background)……………….4 Complete blood cell count (129 Sv/Ev/Tac background)……………….5 Mendelian distribution of RbN750F/0- progeny …………………….82 viii ACKNOWLEDGEMENTS I would like to thank my advisor Jean Y.
Wang for guiding me through this project. I would also like to thank Rimma Levenzon and Irina Hunton for their invaluable help with mouse colony maintenance and tissue dissection; Dr. Kenneth Kaushansky, Dr. Amy Geddes, Dr.
Ian Hitchcock and Norma Fox for characterization of megakaryocyte progenitors; Dr. Nissi Varki and Dr. Gregory Erickson for histological analysis of tissues; Dr. George Widhopf for characterization of lymphocyte population.
And last, but not least, my heartfelt appreciation goes to the past and present members of the Wang lab, whose humor and camaraderie sustained me through many long days and nights. ix VITA Education BS, Cell and Developmental Biology, 1996, University of Rochester, Rochester, NY Graduated cum laude PhD, Biology, 2007 Division of Biological Sciences, University of California, San Diego, La Jolla, CA Thesis advisor: Jean Wang, PhD Honors Bausch and Lomb Science Award (1992) Presidential Academic Fitness Award (1992) Dean's List, University of Rochester (1992-1996) McNair Summer Research Fellowship (1995) Positions Research Fellow, Department of Neurobiology and Anatomy, School of Medicine and Dentistry, University of Rochester, Rochester, NY 1994 Cloned rat neuroretinal cells immortalized with viral A12 protein. Cultured cells in depolarized medium for various periods of time and analyzed expression of A12 using heavy metal marked antibodies and Western Blotting. Research Fellow, Department of Biology, University of Rochester, Rochester, NY 1995-96 Developed and conducted the introduction of and screen for mutations in βν gene of Drosophila integrin using P-element mutagenesis.
Set up crosses, screened progeny, collected embryos and stained them for lac Z expression using antibodies against β- galactosidase. Research Assistant, Research and Development, Invitrogen Corporation, Carlsbad, CA 1996-98 Used standard recombinant DNA techniques to construct 32 vectors, which are currently sold by the company. Scientist II, Department of Genomics, Genos Biosciences, Inc., La Jolla, CA 1998-99 Designed, executed and analyzed all aspects of oncology genomics project including exon trapping, cDNA selection, cDNA library screening, identification of new genes, construction of physical and transcriptional maps, sequencing, mutation detection and bioinformational analysis. x Research Technician III, Department of Chemistry, Laboratory of Peter Schultz, PhD, The Scripps Research Institute, La Jolla, CA 1999-2000 Designed, executed and evaluated experiments to study the effects of small synthetic DNA-binding molecules on transcription of genes in cultured mammalian cells using various promoter constructs fused to luciferase reporter gene.
Studied changes in gene expression using Affymetrix DNA chip technology. Graduate Student with Jean Wang, PhD, Division of Biological Sciences, University of California San Diego, La Jolla, CA 2001-07 Studied the effect of a point mutation in Retinoblastoma gene (Rb-N750F) by generating a knock-in mouse using gene targeting. Performed extensive physiological and histological analysis in order to describe the resulting phenotype, which included elevated levels of platelets and lymphocytes in the peripheral blood of the mutant animals and female sterility due to anovulation. Performed co-immunoprecipitation experiments using 3T3 cells derived from mutant embryos and wild type control littermates to study biochemical properties of the Rb-N750F.
Publications Semenova J, and Zusman S. (1996) “Isolation of Mutations in βν Sub-unit of Integrin in Drosophila Melanogaster”, Proceedings of the Tenth National Conference on Undergraduate Research, vol. Chau NB, Bergseid J, and Wang JYJ (2006). RB and Cancer.
In: Apoptosis and Cancer Therapy, Debatin, K-M., Wiley-VCH, Weinheim, Germany, Chapter 21, pp551-567. Markey MP, Bergseid J, Bosco EE, Stengel K, Xu H, Mayhew CN, Jiang Y, Schwemberger SJ, Babcock G, Jegga AG, Reed MF, Aronow BJ, Wang JYJ, Knudsen ES (accepted for publication to Oncogene). Loss of the Retinoblastoma Tumor Suppressor: Differential Action on Transcriptional Programs Related to Cell Cycle Control and Immune Function. Bergseid J, Jiang Y, Wang JYJ (manuscript in preparation).
The LxCxE binding domain of pRb regulates lymphopoiesis, thrombopoiesis and ovulation. xi ABSTRACT OF THE DISSERTATION Biological Function of the LxCxE-binding Domain of Retinoblastoma Protein by Jacqueline Bergseid Doctor of Philosophy in Biology University of California, San Diego 2007 Professor Jean Y. Wang, Chair Professor Randall S. Johnson, Co-Chair The product of the retinoblastoma gene (pRb) is a tumor suppressor protein that regulates cellular proliferation, apoptosis and differentiation of numerous tissues in mice.
It contains multiple peptide-binding pockets through which it interacts with a host of cellular and viral proteins. The LxCxE-binding pocket of pRb has been highly conserved between pRb proteins from evolutionary distant species; however, the in vivo function of this binding pocket is unknown. The crystal structure of pRB bound to LxCxE peptide was used to design a single point mutation, N757F, which specifically inactivates interactions between pRB and LxCxE-containing proteins without affecting the pRB-E2F interaction. The N750F mutation (analogous to N757F in the human RB) was introduced into the mouse Rb-1 locus by homologous recombination.
The RbN750F/N750F mice do not xii exhibit the phenotype of embryonic lethality observed in Rb-null mice. The pRb-N750F protein does not co-immunoprecipitate with E1A, demonstrating disruption of the LxCxE-binding pocket. The Rb+/- mice develop pituitary tumors with 90% penetrance through LOH. By contrast, the pRb-N750F protein retains its pituitary tumor suppression function as evidenced by the lack of pituitary tumors in RbN750F/N750F and Rb+/N750F mice.
This is consistent with the data from tissue culture experiments demonstrating that RbN750F/N750F fibroblasts do not exhibit any cell cycle defects. The lack of embryonic lethality in RbN750F/N750F mice allowed us to study the effect of this mutation on adult tissues. The RbN750F/N750F mice have elevated levels of platelets and lymphocytes in the peripheral blood. The RbN750F/N750F females are infertile due to anovulation.
These findings demonstrate for the first time that the LxCxE-binding pocket of pRb plays a role in thromobopoiesis, lymphopoiesis and ovulation. The RbN750F/- mice are born at the frequency of 13% and die by the age of 8 months, indicating that, unlike Rb+ allele, the RbN750F allele is haploinsufficient. The RbN750F/- females are also infertile due to the lack of FSH and LH function in the ovaries. xiii CHAPTER I INTRODUCTION Retinoblastoma protein (pRB) was first discovered as the product of a gene whose mutation or loss causes cancer of the retina in children.
Based on statistical analysis of patient data Alfred Knudson proposed a hypothesis that retinoblastoma was caused by two mutational events(30). He also proposed that in the hereditary form of retinoblastoma, the first mutation is inherited from one of the parents and the second mutation occurs in somatic cells, while in the nonhereditary form, both mutations occur in somatic cells. The presence of a germ line mutation in one allele of the RB gene causes a rapid loss of the remaining wild type allele in the retinal cells, leading to the development of bilateral retinoblastoma with multifocal lesions in both eyes before the age of two, with 90% penetrance(34). A spontaneous mutation in one allele of the RB gene that occurs in retinal cells increases the probability of another mutational event in the same cells, resulting in a single tumor in one eye which is observed at an older age.
In addition to retinoblastoma, RB+/- individuals are at increased risk of developing bladder carcinomas, osteosarcomas and fibrosarcomas, indicating that these tissues also contain cell types that are dependent on the presence of functional pRB for tumor suppression(36). Analysis of genomic DNA from affected individuals provided an explanation of molecular mechanism for the development of retinoblastoma(6). Comparison of restriction fragment length polymorphism in DNA derived from normal tissues and 1 2 tumors revealed that wild type chromosome 13 was invariably lost in tumors. The remaining chromosome 13 contained deletions, rearrangements or translocations.
Importantly, none of the seven chromosomes that were also analyzed contained chromosomal mutations, further indicating that gene responsible for the development of retinoblastoma is located on chromosome 13. Subsequent cloning and sequencing of the RB gene opened a possibility to study the mechanism of tumorigenesis at the molecular level(21, 25). Additional evidence that functional pRB is important for tumor suppression came from the findings that several small DNA tumor viruses produce proteins that interact with pRB. Moreover, mutations in viral proteins that abrogated their ability to bind pRB rendered them incapable of transforming cells(8, 24, 39, 41, 46, 48).
Together these findings suggested that oncogenic viruses cause cancer by binding to and inactivating endogenous cellular proteins. Retinoblastoma protein and its functions Human pRB is 928 amino acids long (Figure 1. The protein is divided into four major domains: N, A, B and C. The N-terminus consists of the first 350 amino acids.
Its function is very poorly understood, however, it seems to be dispensable for most of pRB functions(26). The A and B domains contain sequences that are essential for most of pRB functions. These domains are also found in two related proteins, p107 and p130. The A/B domain is highly conserved between all three proteins, which together form a family of “pocket” proteins (reviewed in (12, 27)).