MATRIX ATTACHMENT REGIONS IN THE HUMAN DYSTROPHIN GENE Stephanie Doreen Ditta A tbesis submitted in confordty witb tbe reguirements for the degree of Doctor of Pbilosophy Graduate Department of Molecular and Medical Genetics University of Toronto O Copyright by Stepbanie Doreen Ditta 200 du="-?l?na- Cana The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant à la National Library of Canada to Bibliothèque nationaie du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in microforni, vendre des copies de cette thése sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neitber the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Math Attacbment Regions in the Human Dystrophin Gene Ph.D., 2000 Stephanie Doreen Ditta Department of Molecular and Medical Genetics University of Toronto A bstract Duchenne muscular dystrophy @MD) is a lethal degenerative muscle disorder caused by mutations in the dystrophin gene. Dystrophin is a cytoskeletal protein associated with the plasma membrane. .dystrophin gene is exceptional with mspect CO its size and complexity. It is the largest gene identified to date, containing 79 exons across 2300 kb and at least seven distinct tissue-specific promoters. This X-linked gene is characterized by a high mutation rate, with most affected individuals having chromosomal rearrangernents. Approximately 60% of males with the disease have deietions in the dysimpbin gene and 6% have duplications. In this study, the role of chmatin organization of the dystrophin gene in nuclear functions is investigated. The interactions of DNA with proteins of the nuclear rnatrix rnay be involved in such functions as defining chnimaiin loops or domains, DNA replication, transcriptional reguiation and iiiegitimate recombination. The DNA regions involved in these interactions are specific DNA sequences dedma& attachment regions (MARS). This work examines the possible involvernent of dystrophin gene MARS in both diseasecausing chromosomai rearrangements and transcriptional regulation. Specifically, points of amchment of tbe dystrophin gene to the nuclear matrix have been identifieci in this study at the breakpoints of a duplication hma DMD patient and in the muscle- specfic promoter region. MARS have been identifieci at both the intmn 7 and inîron 9 breakpoints of a dystmphin gene duplication which resulted hm a non-homologous recombination event. This hding indicates the possible positionhg of these breakpoints in close proximity to one another on the nuclear matrïx, proviiàing the opportunity for recombination to occw. Both bceakpoints contain topoisoncerase 1 consensus cleavage sites, suggesting a molecular mechanism for DNA exchange in tbis recombination event. In addition, two MARS have been found to flank the intemal muscle-specific promoter of the dystrophin gene. The proximity of these two MARS to this cis-acting mgulatory element indicates a possible role for these MARS in transcriptional replation hmthis promoter, perhaps through interactions with transcription factors on the nuclear matrix. These results suggest important des for dystrophin gene MARS in both illegitimate recombination and transcriptional regdation in this gene. iii Acknowledgements 1wodd like to thiuik the Canadian Institutes of Health Research (formerly the Medical Research Councii of Canada), tbe Ontario Graduate Scholarship Program and the Hospital for Sick Children Research Training Centre (Toronto) for the financial support of the work ptesented in this thesis. 1 acknowledge my supervisors, Drs. Ronald Worton and Peter Ray, as well as Drs. Brenda Andrews, Paul Sadowski and Lap-Chee Tsui, for serving on my supervisory cornmittee. My thanks to Dr. Arthur Roach for his guidance and support. 1am grateful to Dr. Amira Klip for her kindness, support and valuable advice. 1would like to thank my laboratory colleagues, pst and present, for their help and friendship, especiaily Ramona Cooperstock, Peny Howard, Xiuyuan Hu, Henry Klarnut and Christine Tennyson. 1also thank my friends Carol Freund, Geoff Clarke and Mandy Lowe. My very special thanks to Fîora Krasnoshtein for her constant friendship and encouragement. 1wish to thank my mother, Doreen Ditta, my sisters, Susan Lightstone, Mary Louise Diua and Joan Frawley, and my extended family rnembers for their love and support. Many thanks to my aunt, Jean Dunham, who has made my stay in Toronto enjoyable. Special thanks to my cousins, Cameron Jenkins, who first sparked my interest in genetics, and Kirnberly Gray, who remindecl me of who 1 am. 1 also th& my father, Frank L. Ditta, for al1 that he taught me so long ago. Table of Contents .. Abstract ......................................................................................................u Acknowledgements ........................................................................................iv Table of Contents ........................................................................................ v List of Figures ........................................................................................... vii Chapter 1 - Introduction Overview .................................................................................................... 2 Dystrophin .................................................................................................. -3 Duchenne and Becker Muscular Dystrophy .................................................... 3 Dystrophin Gene ................................................................................... 3 Dystrophin Structure.............................................................................. 6 Dystrophin Function .............................................................................. 8 Dystrophin Isoforms.............................................................................. 9 Matrix Attachment Regions .............................................................................. 12 Nuclear Matrix ................................................................................... -12 MAR Structure.................................................................................... 16 MARS in Replication ............................................................................ -17 MARS in Transcription .......................................................................... 18 MARS in Illegitimate Recombination .......................................................... 20 Rationale .............................................................................................. 21 MARS and Illegitirnate Recombination in the Dystrophin Gene............................ 21 MARS and Transcription in the Dystrophin Gene............................................ 22 Hypothesis ....................................................................................... -24 Chapter 2 .Partid Gene Duplication in the Dystrophin Gene due to Recombination Between Matrix Attachment Regions Abstract ..................................................................................................... 26 Introduction ............................................................................................... -27 Materials and Metbods .................................................................................... 29 Patient ............................................................................................. 29 DNA clones ....................................................................................... 30 Ce11 Culture....................................................................................... 30 in vitro DNA-Binding Assay ................................................................... 30 in vivo MAR Isolation Method ................................................................. 32 Isolation of Total Genomic DNA ............................................................... 33 Results ..................................................................................................... -34 Identification of MAR at intron 9 breakpoint ................................................. 34 Identification of MAR at intron 7 breakpoint ................................................. 40 DNA sequence-based MAR predictions ...................................................... -43 Discussion ............................................................................................. 46 Cbapter 3 .The Role of Matrix Attachment Regions in Transcriptional Regulation fmm the MusckSpecilic Promoter in the Dystrophin Gene Introduction ............................................................................................. -31 Materials and Methods .................................................................................... 53 DNA clones .......................................................................................53 Ce11 Culture ........................................................................................ 54 in vitro DNA-Binding Assay ................................................................ 54 in vivo MAR Isolation Method ................................................................