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Roles of Krüppel Like Factors Klf1, Klf2, and Klf4 in Embryonic Beta-Globin Gene Expression

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Roles of Krüppel Like Factors Klf1, Klf2, and Klf4 in Embryonic Beta-Globin Gene Expression Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 ROLES OF KRÜPPEL LIKE FACTORS KLF1, KLF2, AND KLF4 IN EMBRYONIC BETA-GLOBIN GENE EXPRESSION Yousef Alhashem Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medical Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1880 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Virginia Commonwealth University School of Medicine This is to certify that the thesis prepared by Yousef N. Alhashem entitled “Roles Of Krüppel Like Factors KLF1, KLF2, And KLF4 In Embryonic Beta-Globin Gene Expression” has been approved by the student advisory committee as a satisfactory for the completion of the thesis reqirement for the degree of Master of Science. _____________________________________________ Joyce A. Lloyd, Ph.D., Director of Thesis, School of Medicine _____________________________________________ Rita Shiang, Ph.D., School of Medicine _____________________________________________ David C. Williams Jr., Ph.D., School of Medicine _____________________________________________ Paul B. Fisher, Ph.D., Chair, Department of Human and Molecular Genetics _____________________________________________ Jerome F. Strauss, III, M.D., Ph.D., Dean, School of Medicine _____________________________________________ F. Douglas Boudinot, Ph.D., Dean, Graduate School _____________________________________________ Date © Yousef Nassir Alhashem 2009 ROLES OF KRÜPPEL LIKE FACTORS KLF1, KLF2, AND KLF4 IN EMBRYONIC BETA-GLOBIN GENE EXPRESSION A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University By Yousef Nassir Alhashem, B.Sc. King Saud University, 1999 Director: Joyce A. Lloyd, Ph.D. Professor Department of Human and Molecular Genetics Virginia Commonwealth University Richmond, Virginia June, 2009 II ACKNOWLEDGEMENTS In the first place, I honestly glorify my God for His blessing and help. I would like to thank my parents for their prayer, care, and exceptional support throughout my life. I express my sincere thanks to my wife, Mariam Alhammad for her love and being with me through thick and thin. I would like to thank the Ministry of higher education in Saudi Arabia and the Saudi Arabian Cultural Mission in Washington DC for their financial support and great assistance. I would like to express my sincerest gratitude to my advisor, Dr. Joyce Lloyd for her enthusiasm and patience in guiding me throughout my academic career. This piece of work will never be accomplished without your advice and comments. Your knowledge, insight, and wisdom inspired me and fueled my attitude to pursue my academic goals. I would like to show appreciation to the former and current members in Dr. Lloyd’s lab. Special thanks to Mohua Basu for guiding me during the first days in the lab and helping me thereafter. Special thanks to Dr. Latasha Redmond and Tina Lung for their helpful training and technical support. Great thanks to Aditi Chiplunkar, Christopher Pang, Gabriel Eades, and Divya Vinjamur, for the great help and discussion. I also would like to thank Dr. Gordon Ginder and his lab members for the helpful discussion and using their instruments and reagents. I also thank the lab members of Dr. Taylor and Dr. Moran’s lab for using their instruments. Finally, my best thank goes to my thesis committee members, Dr. Rita Shiang and Dr. David Williams for their precious advice and great insights throughout my Master’s project. III TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................... II TABLE OF CONTENTS ...................................................................................... III LIST OF TABLES ................................................................................................. V LIST OF FIGURES .............................................................................................. VI LIST OF ABBREVIATIONS .............................................................................. VII ABSTRACT ......................................................................................................... XII CHAPTER. I INTRODUCTION ............................................................................ 1 I.1 Erythropoiesis ...................................................................................... 1 I.1.1 Primitive Erythropoiesis ...................................................................... 2 I.1.2 Transcriptional regulation of erythropoiesis ........................................ 4 I.1.3 The β-globin locus ................................................................................ 6 I.1.4 β-globin-related hemoglobinopathies ................................................. 10 I.2 Krüppel Like Factors ......................................................................... 12 I.2.1 Krüppel Like Factor 1 ....................................................................... 17 I.2.2 Krüppel Like Factor 2 ....................................................................... 24 I.2.3 KLF1 and KLF2 have compensatory roles in primitive erythropoiesis 32 I.2.4 Krüppel Like Factor 4 ....................................................................... 33 IV I.2.5 KLF1, KLF2, and KLF4 may have functional redundancy in the activation of β-like globin genes ........................................................ 37 CHAPTER II MATERIALS, METHODS, AND RESULTS .............................. 39 II.1 Material and Methods ........................................................................ 39 II.1.1 Generation and dissection of triple knockout mice ............................ 39 II.1.2 Genotyping ......................................................................................... 41 II.1.3 RNA preparation and cDNA synthesis .............................................. 43 II.1.4 Quantitative RT-PCR ......................................................................... 44 II.1.5 Statistical analysis ................................................................................ 46 II.2 Results and Discussion ....................................................................... 46 II.2.1 KLF1 does not regulate KLF2 in blood cells ..................................... 46 II.2.2 Triple knockout embryos are alive at day 9.5 .................................... 48 II.2.3 Roles of KLF1, KLF2, KLF4 on βh1- and Ey-globin gene expression: 49 CHAPTER III DISCUSSION AND FUTURE DIRECTIONS ............................ 53 III.1 Limitations of the study ..................................................................... 56 III.2 Future Directions ................................................................................ 57 LIST OF REFERENCES ...................................................................................... 62 VITA ..................................................................................................................... 72 V LIST OF TABLES Table 1 : Primer sets for genotyping of mouse KLF1, KLF2, and KLF4............. 42 Table 2: Probe and Primer sets for qRT-PCR ....................................................... 45 Table 3: Number of live E9.5 embryos obtained from the timed mating ............. 49 VI LIST OF FIGURES Figure 1: The spatial/temporal expression of α- and β- globin loci in human. ....... 7 Figure 2: Temporal expression of the β-globin gene in the mouse......................... 8 Figure 3: Illustration of a KLF protein. ................................................................ 13 Figure 4: Representation of the zinc finger domains in the human KLFs ............ 14 Figure 5: Phylogenetic analyses of the KLFs ....................................................... 15 Figure 6: schematic representation of WT and Knockout alleles of KLF1, KLF2, and KLF4 genes ............................................................. 40 Figure 7: Representation of gel electrophoresis for KLF1, KLF2, and KLF4 genotyping .................................................... 42 Figure 8: KLF2 expression in KLF1 knckout mice at E9.5. ................................. 47 Figure 9: Expression levels of the mouse βh1 mRNA at E9.5 in different KLF1, KLF2, and KLF4 genotypes. .................................. 50 Figure 10: Expression levels of the mouse Ey mRNA at E9.5 in different KLF1, KLF2, and KLF4 genotypes. .................................. 51 VII LIST OF ABBREVIATIONS % ............................. percent α .............................. Alpha β .............................. Beta βh1 .......................... beta-like embryonic chain (mouse hemoglobin Z) βLCR ....................... β-globin Locus Control Region βmaj .......................... beta major globin βmin .......................... beta minor globin ............................... Gamma δ ............................... Delta ............................... Epsilon δ ............................... Zeta θ ............................... Theta .............................. micro g ............................ micro-gram l ............................. micro-Liter χ2 ............................. chi-square 2D ............................ two-dimensional
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