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Mixed Lineage Luekemia Histone Methyltransferases MIXED LINEAGE LUEKEMIA HISTONE METHYLTRANSFERASES IN HORMONAL REGULATION OF HOXB9 AND TARGET GENE REGULATION by BISHAKHA SHRESTHA Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON December 2011 ACKNOWLEDGEMENTS First and foremost, I would sincerely like to thank my graduate advisor Dr. Subhrangsu S. Mandal for his immense encouragement and support, for his patience and for his helpful guidance to develop my scientific knowledge without whom my pursuit of a Doctoral degree would have been almost impossible. I would also like to thank Dr. Jongyun Heo and Dr. Roshan Perera, my committee members for their encouragement, guidance and immense support. I would like to thank the Chemistry Department of University of Texas at Arlington for providing me the grounds to pursue my higher education. I am also thankful to all the faculty and staff at Chemistry Department who have helped me in my graduate student career in many different ways. I would like to thank Dr. Khairul Ansari, our postdoctoral associate for his help with the experiments and for sharing his scientific and technical knowledge. Also, sincere thanks to my former and current lab members: Dr. Getachew Woldemariam, Dr. Bibhu P. Mishra, Sahba Kasiri, Imran Hussain, Arunoday Bhan and all the other lab members for help with experiments and providing moral support. I would also like to thank all my friends in the DFW area and all those who lent out a helping hand and provided kind and encouraging words. ii My heartfelt thanks go out to my parents and my family who had immense faith in my capabilities and provided my support all along. Thank you so much for always being there and for your selfless and unconditional love. November 21, 2011 iii ABSTRACT MIXED LINEAGE LUEKEMIA HISTONE METHYLTRANSFERASES IN HORMONAL REGULATION OF HOXB9 AND TARGET GENE REGULATION Bishakha Shrestha, PhD The University of Texas at Arlington, 2011 Supervising Professor: Subhrangsu S. Mandal Homeobox gene (HOX) genes are evolutionary conserved genes that play important roles in cell differentiation, cell proliferation and embryogenesis. HOX genes bind to the DNA via their homeodomain and act as transcription factors. Of the 39 HOX genes present in human, HOXB9 is known to be a critical player in skeletal and mammary gland development. HOXB9 also regulates renin gene expression which is a critical player in Renin-Angiotensin system. Recent studies also demonstrate that HOXB9 is critical for angiogenesis. I have investigated transcriptional regulation of HOXB9 and its potential biochemical function during cell cycle progression and tumorigenesis. My studies demonstrate that HOXB9 is an estrogen responsive gene. HOXB9 promoter contains multiple estrogen response elements through which they interact with estrogen-receptors and regulate gene expression in presence of estrogen. Mixed lineage leukemia (MLL) family of histone methylases that are key players in iv gene activation and epigenetics, coordinate with estrogen-receptors during transcriptional activation of HOXB9 in presence of estrogen. Studies also demonstrate that HOXB9 is overexpressed in breast cancer. HOXB9 regulates various cell cycle regulatory genes that includes various Cyclins and p-proteins and regulates cell cycle progression. Overexpression of HOXB9 induces cell cycle arrest in G0/G1 phase and ultimately induces apoptosis. Homeodomain of HOXB9 plays critical roles in transcriptional regulation of cell cycle regulatory genes and cell cycle progression. Further studies demonstrated that HOXB9 overexpression stimulates three-dimensional growth of tumor in colony formation assay. HOXB9 controls the expression of various tumor growth and angiogenic factors via involvement of its homeodomain and thus influences tumor growth. In conclusion, HOXB9 is an estrogen responsive gene and is overexpressed in breast cancer. HOXB9 is a crucial player in cell cycle regulation and tumorigenesis. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................ ……………..ii ABSTRACT.................................................................................................................................iv LIST OF ILLUSTRATIONS ..........................................................................................................ix LIST OF TABLES ....................................................................................................................... xii LIST OF ABBREVIATIONS ...................................................................................................... xiii Chapter Page 1. MIXED LINEAGE LEUKEMIAS IN HISTONE METHYLATION, GENE EXPRESSION, AND HORMONE SIGNALLING …………………..………..….. ..... .1 1.1 Introduction .................................................................................................. 1 1.2 Histone modification and histone code hypothesis ....................................... .2 1.3 MLLs are H3K4 specific histone methyl transferases ................................... .4 1.4 MLLs contain various functional domains..................................................... .6 1.5 MLL1 is rearranged in leukemia ................................................................... .9 1.6 MLLs exist as multi-subunit complexes ...................................................... .10 1.7 MLLs are master regulator of HOX genes .................................................. .12 1.7.1 HOX gene structure and function ................................................ 12 1.7.2 HOX genes and its interacting partners ....................................... 14 1.7.3 Specificity in HOX target gene regulation .................................... 15 1.7.4 HOX gene and its downstream target genes ............................... 16 1.8 Endocrine regulation of HOX genes and its downstream target genes ....... .18 1.8.1 Retinoic acid and its influence on HOX gene expression ............. 18 vi 1.8.2 Estrogen and its influence on HOX gene expression .................. 19 1.8.3 Progesterone and its influence on HOX gene expression ............ 20 1.8.4 Testosterone and its influence on HOX gene expression ............. 21 1.9 Discussion ................................................................................................ .22 2. HISTONE METHYLASE MLL1 AND MLL3 COORDINATE WITH ESTROGEN RECEPTORS IN ESTROGEN MEDIATED HOXB9 EXPRESSION ............................... 24 2.1 Introduction ................................................................................................ 24 2.2 Materials and methods ............................................................................... 26 2.2.1 Cell culture, estrogen treatment and antisense mediated knockdown experiments ...................................................................... 26 2.2.2 RNA Extraction, Reverse Transcription and Real Time PCR ....... 27 2.2.3 Protein extraction and Western blotting ....................................... 28 2.2.4 Chromatin Immuno-precipitation (ChIP) experiment .................... 28 2.2.5 Luciferase reporter assay ............................................................ 29 2.3 Results ....................................................................................................... 30 2.3.1 Effects of estrogen (E2) on HOXB9 gene expression .................. 30 2.3.2 HOXB9 promoter contains potential estrogen response elements ...................................................... 32 2.3.3 Estrogen receptors play critical roles in E2-mediated transcriptional activation ............................................................. 37 2.3.4 ER α and ER β bind to HOXB9 promoter in presence of E2........... 39 2.3.5 Roles of MLLs in E2 mediated regulation of HOXB9 .................... 42 2.3.6 MLL histone methylases bind to HOXB9 promoter in presence of E2 ........................................................ 44 2.4 Discussion ................................................................................................. 47 3. HOMEODOMAIN CONTAINING PROTEIN HOXB9 PLAYS CRITICAL ROLES IN CELL CYCLE PROGRESSION, TUMORIGENESIS, AND IS OVEREXPRESSED IN BREAST CANCER ................................................................................................... 52 3.1 Introduction ................................................................................................ 52 vii 3.2 Experimental section .................................................................................. 53 3.2.1 Cell culture and cell synchronization ........................................... 53 3.2.2 Immuno-histological analysis of breast cancer tissue microarray. ............................................. 54 3.2.3 Establishment of HOXB9 (full length) and HOXB9 homeodomain stably transfected cell line. ..................... 55 3.2.4 Antisense mediated knockdown experiments .............................. 55 3.2.5 RNA extraction, reverse transcription and real time PCR ...................................................................... 56 3.2.6 Protein extraction and western blotting ........................................ 57 3.2.7 Chromatin immuno-precipitation assay ........................................ 57 3.2.8 Immuno-fluorescence microscopy ............................................... 58 3.2.9 FACS analysis ............................................................................ 58 3.2.10
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