UNIVERSITY OF CALIFORNIA Los Angeles Dissecting transcriptional control by Klf4 in somatic cell reprogramming A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biological Chemistry by Huajun Zhou 2017 ABSTRACT OF THE DISSERTATION Dissecting transcriptional control by Klf4 in somatic cell reprogramming by Huajun Zhou Doctor of Philosophy in Biological Chemistry University of California, Los Angeles, 2017 Professor Gregory S. Payne, Chair Ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, coverts somatic cells directly into induced pluripotent stem cells (iPSCs), which are functionally equivalent to embryonic stem cells (ESCs). The discovery of iPSC has been reshaping the methodology of disease modeling and drug screening in the past decade, and provides tremendous promise for regenerative medicine. However, the mechanism underlying this conversion process, reprogramming, is not yet fully understood. I aimed to dissect the reprogramming process, by characterizing the functional domains of one reprogramming factor Klf4. The transcriptional activation domain (TAD) of Klf4 was revealed to be critical for reprogramming. To search for the factors that mediates the functionality of Klf4 TAD, I identified transcriptional coactivators CBP/p300 and Mediator complex as the physical interaction partners of Klf4 TAD, and further showed that this interaction is functionally required ii for Klf4 mediated transcriptional activation in reprograming. Clathrin heavy chain, initially identified as a physical interaction partner of Klf4 TAD, was shown to be not required for Klf4 transcriptional activation. Clathrin heavy chain was furthered characterized for its potential transcriptional activation activity in CHC-TFE3, a chromosomal fusion discovered in renal cell carcinoma. Clathrin heavy chain, as a conventional vesicle coat protein involved cellular membrane trafficking, was further studied with its accessory protein Irc6 in budding yeast. iii The dissertation of Huajun Zhou is approved. Michael F. Carey Siavash K. Kurdistani Esteban C. Dell'Angelica Kathrin Plath Gregory S. Payne, Committee Chair University of California, Los Angeles 2017 iv This work is dedicated to my parents. Thank you for your love and support. v TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................... vi FIGURES AND TABLES ........................................................................................................... viii ACKNOWLEDGEMENTS ........................................................................................................... xi VITA ............................................................................................................................................ xiii CHAPTER 1 ................................................................................................................................... 1 Abstract ........................................................................................................................... 2 The discovery of iPSC .................................................................................................... 2 Nuclear reprogramming .................................................................................................. 4 Molecular mechanisms of reprogramming ..................................................................... 5 Epigenetic changes in reprogramming ............................................................................ 8 Overview of Oct4, Sox2, and Klf4 ............................................................................... 14 Molecular mechanism of transcriptional control by Oct4, Sox2, and Klf4 .................. 16 Concluding remarks ...................................................................................................... 18 References ..................................................................................................................... 19 CHAPTER 2 ................................................................................................................................. 28 Abstract ......................................................................................................................... 29 Introduction ................................................................................................................... 29 Results ........................................................................................................................... 31 Discussion ..................................................................................................................... 40 Contributions ................................................................................................................ 43 Materials and Methods .................................................................................................. 44 References ..................................................................................................................... 83 vi CHAPTER 3 ................................................................................................................................. 88 Abstract ......................................................................................................................... 89 Introduction ................................................................................................................... 89 Results ........................................................................................................................... 95 Discussion ................................................................................................................... 100 Materials and Methods ................................................................................................ 103 References ................................................................................................................... 121 CHAPTER 4 ............................................................................................................................... 126 Abstract ....................................................................................................................... 127 Introduction ................................................................................................................. 127 Results ......................................................................................................................... 129 Discussion ................................................................................................................... 134 Materials and Methods ................................................................................................ 137 References ................................................................................................................... 155 CHAPTER 5 ............................................................................................................................... 157 Functional significance of CBP/p300 in H3K27ac domain establishment ................. 158 Structural role of Mediator in 3D organization of genome ......................................... 159 Interwoven relationships of multiple factors in enhancer-promoter interaction and transcriptional activation ......................................................................................................... 160 Concluding remarks .................................................................................................... 161 References ................................................................................................................... 162 vii FIGURES AND TABLES Figure 2-1 Klf4 regions important for reprogramming. ................................................................ 56 Figure 2-2 TAD sequence requirements for transcriptional activation and reprogramming. ....... 58 Figure 2-3 Proteins bound differentially to wild-type TAD compared to reprogramming defective mutants. ......................................................................................................................................... 60 Figure 2-4 Direct and functional interactions of p300/CBP with Klf4 TAD. .............................. 62 Figure 2-5 Direct and functional interactions of Mediator with Klf4 TAD. ................................ 64 Figure 2-6 Genome-wide gene expression effects of wild-type and I106A Klf4 TAD. .............. 66 Table 2-1 Number of genes significantly up- or down- regulated in cells expressing full-length Klf4 (FL), wild-type (WT) or I106A TAD fused to Klf4 aa210-483 compared to cells expressing tdTomato (TOM) or WT compared to I106A. Significance determined as described in Methods. ....................................................................................................................................................... 68 Figure 2-S1 Characterization of Klf4 constructs. ......................................................................... 69 Figure 2-S2 Klf4 TAD transcriptional activation activity. .......................................................... 71 Figure 2-S3 Binding of CBP/p300 domains to Klf4 TAD and overexpression of CBP and p300. ....................................................................................................................................................... 73 Figure 2-S4 Purified Mediator complex. .....................................................................................