University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 2000 Regulation of skeletal muscle differentiation by ATBF1, a multiple homeodomain-zinc finger transcription factor Berry, Fred Brandon Berry, F. B. (2000). Regulation of skeletal muscle differentiation by ATBF1, a multiple homeodomain-zinc finger transcription factor (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/23689 http://hdl.handle.net/1880/39899 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca NOTE TO USERS Page(s) not included in the original manuscript and are unavailable from the author or university. The manuscript was microfilmed as received. This reproduction is the best copy available. THE UNIVERSITY OF CALGARY Regulation of Skeletal MuscIe Differentiation by ATBF1, a Multiple Homeodomain-zinc Finger Transcription Factor Fred Brandon Berry A DISSERTATION SUBMITTED TO THlE FACC?TYOF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY CALGARY, ALBERTA APRIL, 2000 O Fred Brandon Berry 2000 National Library Bibliotheque nationale I+IofCanada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wetlington Street 395. rue Wellington Ottawa ON KIA ON4 Ottawa ON KIA ON4 Canada Canada Your fib Worm rdfarmce Our fiIe Ndrro fel(Irenc8 The author has granted a non- L'auteur a accorde me licence non exclusive licence allowing the exclusive permeftant a la National IArary of Canada to Bibliotheque nationale du Canada de reproduce, loan, distribute or sell reproduire, prster, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/fih, de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la propriiete du copyright in this thesis. Neither the droit d'auteur qui protkge cette these. thesis nor substantial extracts from it Ni la these ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent Etre knprimks reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract The ATBFl gene encodes two protein isoforms, the 404 kDa ATBF1-A, possessing four homeodomains and 23 zinc fmgers, and the 306 kDa ATBF1-B, lacking a 920-amino acid N-terminal region of ATBFI-A. We found that ATBF1-A was expressed in proliferating CZC 12 myoblasts but its expression levels decreased upon induction of myogenic differentiation in low serum medium. This down- regulation of ATBF la is one of the requirements for muscle differentiation. Cells constitutively expressing ATBF1 -A fail to differentiate as characterized by aberrant expression patterns of myogenic regulatory transcription factors, cell cycle regulatory proteins, and muscle structural proteins. In addition, these cells were characterized by high levels of expression of Id3 arid cyclin D 1, known to inhibit C2C 12 dBerentiation. In contrast, transfection of C2C 12 cells with the ATBF 1-B isoform resulted in an acceleration of myogenic differentiation, as indicated by an earlier onset of MHC expression and the formation of a higher percentage of multinucleated myotubes. In 10T1/2 cells, transfection of ATBF 1-A prevented MyoD-dependent myogenic conversion, whereas ATBF 1B enhanced this process. In addition this study identified the mouse Mm4 gene as a target of ATBF 1 regulation. A region of ATBFl-A containing homeodomain 4 interacted with an AT-rich element overlapping the El E-box of the MRF4 promoter and displaced myogenin from this El site. To inhibit the MyoD-dependent activation of the MRF4 promoter by ATBF 1-A, the binding to these sites was not sufficient, but two iii segments of the ATBF1-A-specific N-terminal region were also required. We show that the ATBF1-A-specific regions possess transcription repressor activity. These results show that the ATBFl isoforms regulate myogenesis in an opposite manner. The down-regulation of ATBF 1-A expression occurs in response to the removal of growth factors from the mediun and is a prerequisite to initiate terminal differentiation of C2C 12 cells. ATBF 1-A-expressing cells remain undifferentiated but enter a reversible quiescence in response to DM, which is a feature common to muscle satellite and reserve cells. Therefore, ATBFl-A may divert myoblasts from entering terminal differentiation and may promote &se cells into adopting a reserve cell identity. Acknowledgments I wish to express my gratitude to my supervisor. Dr. Taiki Tarnaoki for his support, patience and encouragement throughout the duration of this project. I thank him for the opportunity to undertake this research. allowing me the fieedom to explore the topics in which I was interested, and for stressing the foundations for good scientific research. I would also like to extend my appreciation to the members of my supervisory committee. Dr. Gilbert Schultz, Dr. Frans van der Hoorn and Dr. Henk Zwien. for their helpfd comments and suggestions. I would like to thank my lab members, both past and present. Dr. Yutaka Miura. Dongping Ma. Dr. Koichiro Mihara, Dr. Petr Kaspar, and Dr. Kazuo Fushimi for their helpful discussions and assistance. 1 thank Howard Chen for his technical assistance. I am grateful for the many friends that I have made throughout the duration of my stay in Calgary. In particular I thank Robbie Loewith and Chris Howlett for their practical applications of yeast. I extend my thanks to my family for their continuous support. Finally to Misbah Qureshi, I am indebted to her for her companionship and encouragement. I would like to acknowledge financial support provided through Faculty of Graduate Studies Research Scholarships and from grants awarded to my supervisor from the National Cancer Institute of Canada. Table of Contents Approvalpage ................................................... ii Abstract ...................................................... iii AcknowIedgments ................................................ v Table of Contents ............................................... vi ListofFigures ................................................... x ListofTables .................................................. xii List of Abbreviations ............................................ xiii 1. Introduction .................................................. 1 1.1 ZFHproteins regulate cellular development and differentiation...... 2 1.1.1 Class I ZFH proteins ............................... 2 1.1.2 Class II ZFHproteins ............................... 4 1 -2ATBF 1 is a zinc-finger homeodomain transcription factor .......... 5 1 .2.1 ATBF 1 structural-function relationships ................. 6 1.2.2 Comparison between ATBF1-A and B isoforms......... 10 1.2.3 The homeodomains .............................. 11 1.2.4 The zinc-fmgers .................................. 13 1.3 Potential functions for ATBF 1 in embryonic development......... 14 1.4 Regulation of vertebrate skeletal muscle differentiation........... 17 1.4.1 The myogenic differentiation program ................ 18 1.4.2 Induction of MRF expression ....................... 20 1.4.3 Molecular mechanisms of MRF activity................ 22 1.4.4 Integration of muscle differentiation and cell cycle regulation. ............................................. 26 1.5 Rationale for current research .............................. 29 2 . Materials and Methods ........................................ 32 2. 1Plasmids............................................... 32 2.2 Cell Cultures and Stable Transfections........................ 32 2 -3 Southern Blotting ........................................ 35 2.4 mRNA expression analysis ................................. 37 2.4.1 RNA Isolation................................... 37 2.4.2 RNase protection assays ........................... 38 2.4.3 Northern Blotting ................................ 39 2.5 Irnrnunocytochemistry. ................................... 40 2 -6 BrdU incorporation ...................................... 41 2.7 Western Blotting........................................ 42 2.8 Myogenic conversion assays ................................ 44 2 -9 Luciferase reporter assays .................................. 44 2.10 Mammalian one-and two-hybrid assays ...................... 45 2.1 1 DNA-protein interactions................................ 46 2.1 1.1 Nuclear Extract Isolation .......................... 46 2.1 1.2 Probe preparation............................... 47 2.11.3 In vitro translation of proteins ...................... 48 2.1 1.4 Bacterially expressed proteins....................... 48 2.11 -5 ElecQophoretic Mobility Shift Assays (EMSAs) .......... 49 2.1 1.6 W cross-linking of proteins to nucleic acids........... 49 2.12 Immunoprecipitation and His-tag pull-down assays ............. 50 2.13 Tibialis anterior regeneration ............................. 52 3.1 Expression of ATBFI -A during myogenic differentiation.......... 53 3.2 ATBF 1-A inhibits myogenic differentiation of C2C 12 cells ........ 57 3 -3 ATBF 1-A expressing C2C12