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Analysis of Long Range Gene Regulation in Drosophila; Insights Analysis Of Long Range Gene Regulation In Drosophila; Insights From The dPax2 sparking Enhancer by Nicole C. Evans A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cell and Developmental Biology) in the University of Michigan 2012 Doctoral Committee: Associate Professor Scott Barolo, Chair Professor Kenneth Cadigan Assistant Professor Catherine Collins Assistant Professor Kentaro Nabeshima DEDICATION To my family, who have been unfailingly supportive through my entire education and the doctoral experience. My Grandpa Vern, who assigned my sister and me our future careers at age ten, but would have loved me just the same if I had not followed his instructions. To my Grandma Judy, who can make anything positive and has always been there for me with her love and encouragement. To my sister Katelyn, whose sense of humor and sassiness makes my world bright. Especially to my parents, my Dad who taught me to approach every problem with a creative and open mind and my Mom who inspired me to learn and teach others. Without their laughter, love, and support I would not be here chasing my dreams. ii ACKNOWLEDGMENTS I deeply thank Christina Swanson for her friendship and enabling me to pursue a study of the sparking enhancer. I am grateful to Amy Strom and Alex Chappell whose smiling faces and eager brains remind me why I love science every day, for their contributions to this work, and for all the mini preps. iii TABLE OF CONTENTS DEDICATION………..…………………………………………………………..………ii ACKNOWLEDGEMENTS………………………….…………………..……………...iii LIST O F FIGURES…………..……………………………………………………….xiii LIST OF TABLES……………………………………………………………………..xvii CHAPTER 1 SPARKLING INSIGHTS INTO ENHANCER, STRUCTURE, FUNCTION, AND EVOLUTION ............................................................................ 1 1.1 The enhancer: what is it and what can it do? .......................................... 1 1.1a How do enhancers activate gene expression? ................................. 3 1.1b Combinatorial inputs determine transcriptional output ...................... 4 1.1c Models of enhancer structure ........................................................... 5 1.1d Persistent questions in the field ........................................................ 8 1.2 The sparkling enhancer of the dPax2 gene ............................................. 9 1.2a sparkling: enhanceosome or billboard? .......................................... 16 1.2b Combinatorial control of sparkling’s activity .................................... 20 1.2c Structural and sequence constraints channel sparkling output ....... 24 1.2d Low-affinity binding sites and the Notch response .......................... 26 1.3 sparkling in relation to the study of gene regulation .............................. 27 iv 1.4 Acknowledgements ............................................................................... 30 1.5 References ............................................................................................ 30 CHAPTER 2 LONG RANGE GENE REGULATION AND THE IDENTIFICATION OF THE SPARKLING RCE ....................................................... 36 2.1 Abstract ................................................................................................. 36 2.2 Introduction ............................................................................................ 38 2.2a Looping ........................................................................................... 42 2.2b Linking ............................................................................................ 45 2.2c Tracking .......................................................................................... 47 2.2d Facilitated tracking .......................................................................... 49 2.2e Change in nuclear localization ........................................................ 51 2.2f Generation of noncoding RNAs ...................................................... 54 2.2g The dPax2 sparkling enhancer as a model distally located enhancer …………………………………………………………………………….59 2.3 Results .................................................................................................. 63 2.3a Evidence for a special type of regulatory site, specifically mediating action at a distance ..................................................................................... 63 2.3b sparkling regions 5 and 6 are required more for initiation than maintenance of gene expression ................................................................. 65 2.3c sparkling region 5 contains sequences necessary for enhancer repression in photoreceptors ....................................................................... 66 v 2.3d The known transcription factor binding sites and spa region 4 are crucial for robust enhancer activity .............................................................. 68 2.4 Discussion ............................................................................................. 70 2.4a sparkling regulatory sequences each contribute unique roles to enhancer function ........................................................................................ 71 2.4b Functional evidence for a special enhancer regulatory element, mediating remote interactions but not patterning ......................................... 73 2.5 Experimental methods ........................................................................... 75 2.5a Enhancer constructs ....................................................................... 75 2.5b Mutagenesis by overlap extension PCR (Sewing PCR) .................. 75 2.5c Assembly PCR ................................................................................ 76 2.5d Enhancer cloning, vectors, and transgenesis ................................. 76 2.5e Tissue preparation, staining, and microscopy ................................. 77 2.6 Acknowledgments ................................................................................. 77 2.7 References ............................................................................................ 77 CHAPTER 3 STRUCTURE FUNCTION ANALYSIS OF THE SPARKLING REMOTE CONTROL ELEMENT ........................................................................ 82 3.1 Abstract ................................................................................................. 82 3.2 Introduction ............................................................................................ 84 3.3 Results .................................................................................................. 88 3.3a Analysis of sparkling in multiple insertion contexts ......................... 88 vi 3.3b The effect of position and copy number on RCE activity ................. 98 3.3c DNA sequences other than the RCE can convey long-range activity on sparkling ............................................................................................... 102 3.3d Analysis of sparkling’s promoter preferences ............................... 105 3.4 Discussion ........................................................................................... 111 3.4a Observations on sparkling and promoter regulation...................... 113 3.4b Mechanisms of sparkling action .................................................... 117 3.4c Identification of putative protein binding sites that may facilitate RCE activity..…………………………………………………………………………..120 3.5 Experimental methods ......................................................................... 124 3.5a Vector construction, reporter gene generation, and transgenesis . 124 3.5b Tissue preparation, antibody staining, microscopy ....................... 125 3.5c Quantification of GFP expression ................................................. 126 3.5d DNA motif analysis ........................................................................ 127 3.6 Acknowledgments ............................................................................... 127 3.7 References .......................................................................................... 127 CHAPTER 4 A POTENTIAL ROLE FOR THE SIX FAMILY MEMBER, SINE OCULIS, IN SPARKLING ACTIVITY ................................................................ 133 4.1 Abstract ............................................................................................... 133 4.2 Introduction .......................................................................................... 135 4.3 Results ................................................................................................ 141 vii 4.3a Sine oculis interacts with the sparkling RCE in vitro ..................... 141 4.3b sparkling region 4 can convey RCE activity which is potentially mediated by Sine oculis............................................................................. 144 4.3c sparkling activity requires specific DNA sequence input in the position of region 4 .................................................................................... 149 4.3d Sine oculis interacts with spa region 5 possibly through a homeodomain binding site......................................................................... 156 4.3e sparkling regions 5 and 6 can also activate gene transcription at a distance ..................................................................................................... 161 4.4 Discussion ........................................................................................... 163 4.4a Both strict
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