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Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene

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Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1995 Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene. Haiqing Fu Schiltz Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Schiltz, Haiqing Fu, "Transcriptional Studies of the Muscle-Specific Expression of the Rabbit Muscle Phosphofructokinase Gene." (1995). LSU Historical Dissertations and Theses. 6075. https://digitalcommons.lsu.edu/gradschool_disstheses/6075 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. Hie quality of this reproduction Is dependent upon the quality of the copy snbmitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandardm a rg in * , and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are mi«ing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. 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A Beif & Hewed information Company 300 Norm Zeeb Road Ann Arbor Ml 48106-1346 USA 313-761-4700 800 521-0600 TRANSCRIPTIONAL STUDIES OF THE MUSCLE-SPECIFIC EXPRESSION OF THE RABBIT MUSCLE PHOSPHOFRUCTOKINASE GENE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biochemistry by Haiqing Fu Schiltz B.S., South China Normal University, P. R. C., 1983 M.S., Zhongshan University, P. R. C., 1986 December 1995 UMI Numbert 9613427 UMI Microform 9613427 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, Ml 48103 ACKNOWLEDGMENTS I would like to express my sincere thanks to all of those whose support and encouragement have helped me during my graduate school career. I thank my mentor, Dr. Simon H. Chang, who has provided professional advice and financial support. I also thank the members of my advisory committee, Dr. Eric Achberger, Dr. Sue Bartlett, Dr. Ding Shih, and Dr. Patrick DiMario for their guidance and advice. I thank the Department of Biochemistry for providing me the teaching assistantship. I would also like to extend my thanks to my fellow graduate students in Dr. Chang's lab and in the department for all the wonderful memories that we have shared. Special thanks to Drs. Harold Weintraub of the Fred Hutchinson Cancer Research Institute in Seattle, Victor Lin of the University of Texas, Southwestern Medical Center in Dallas, and Hans Arnold of the University of Hamburg in Germany, for providing me the myogenic cDNA expression vectors that have been very important for this work. I dedicate this dissertation to Dr. Harold Weintraub who died of a brain tumor in early March of 1995. He will inspire me for years to come. I am specially grateful to my parents, my brother Hai-Shan and his wife Xiao-Yun, and my relatives in China whose love, support, and understanding made it possible for me to fulfill my dream. I also thank my mother-in-law Mrs. Tosso and her family, my father-in-law Mr. Schiltz, and his wife Sally for their love and support. Most of all, I thank my husband Louie for his love, patience, advice, inspiration, and sacrifice that have carried me through this journey. We are very grateful for our three-year old son Stephen who has brought us joy, love, and hope. ii TABLE OF CONTENTS ACKNOWLEDGMENTS.............................................................................................. ii LIST OF FIGURES...........................................................................................................v LIST OF ABBREVIATIONS vi i ABSTRACT .......................................................................................................................ix CHAPTER 1 LITERATURE REVIEW ........................................................................... 1 1.1 Enzyme Kinetic Properties and Physiological Relevance of PFK.................................................. 1 1.2 Tissue-Specific Distribution of PFK Isozymes and Their mRNAs ........................................................4 1.3 Structural Organization of the PFK Genes ...................................6 1.4 Transcriptional Control of the Muscle PFK Genes ................. 11 1.5 Control Mechanisms of Muscle Gene Expression ...................13 1.6 Structure, Function, and Regulation of the Myogenic bHLH Proteins ................................................ 16 1.7 Other Muscle-Specific Regulatory Factors ................................. 30 1.8 Gene Tissue-Specific Inactivation by DNA Methylation .....................................................................33 1.9 Tissue-Specific Expression of the L- and C-PFK Genes. ..............................................................35 1.10 Research Objectives............................................................................36 2 MATERIALS AND METHODS.......................................................... 39 2.1 M a te ria ls................................................................................................39 2.1.1 Enzymes and Sera .....................................................................39 2.1.2 Chemicals and Culture Media ............................................. 39 2.1.3 Cell Lines, Bacterial Strains, and Helper Phage ..............41 2.1.4 DNA Plasmids ..........................................................................41 2.1.5 Radioisotopes and Oligonucleotides ................................. 43 2.2 Methods ................................................................................................. 43 2.2.1 Construction of PFK-CAT Chimeras ................................. 43 2.2.2 Cell Cultures .............................................................................. 52 2.2.3 Transfection and Enzymatic Assays ................................... 55 2.2.4 Preparation of Glutathione S-transferase Fusion Proteins .................................................................... 57 2.2.5 Preparation of Nuclear Extracts ........................................... 58 2.2.6 l32PJ End-Labeling of DNA Probes .......................................59 2.2.7 Electrophoretic Mobility Shift Assay (EMSA) .................60 2.2.8 DNasel Footprinting ............................................................... 61 3 RESULTS AND DISCUSSION ............................................................ 63 3.1 Transactivation of the RM-PFK Gene by the Myogenic bHLH Factors............................................................... 63 3.2 Delineation of the Positive and Negative Regulatory Elements within the RM-PFK 5'-Flanking Region ........... 76 3.3 Interactions of MyoD and the C2C12 Nuclear Proteins with the El Enhancer In V itro.................................84 3.4 Determination of the Boundary and Activity of the Proximal Promoter ...........................................................93 3.5 Detection of Multiple Protein Binding Sequences within the Proximal Promoter Region .................................106 3.6 Characterization of Another Muscle-Specific Promoter ......................................................... 116 4 SUMMARY, CONCLUSIONS, AND FUTURE STUDIES..............................................................122 4.1 Summary and Conclusions ...........................................................122 4.2 Future Studies ................................................................................... 128 REFERENCES................................................................................................................131 APPENDIXES A RESTRICTION ENDONUCLEASE DIGESTION MAP OF THE 3 KB 5’ FLANKING SEQUENCE OF THE RM-PFK GENE.................................................................151 B LETTERS OF PERMISSION ................................................................ 160 VITA ................................................................................................................................. 166 i v LIST OF FIGURES 1-1 Schematic diagram of the exon-intron composition of the rabbit muscle
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