BTB domain dimerization: Development of a protein-protein interaction assay By Qingniao (Carol) Wang A thesis submitted in conformity with the requirement for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Qingniao (Carol) Wang (2009) BTB domain dimerization: Development of a protein-protein interaction assay Qingniao (Carol) Wang Masters of Science 2009 Department of Biochemistry University of Toronto Abstract In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies. ii Acknowledgements This research project would not have been possible without the support of many people. I wish to express my gratitude to my supervisor, Dr. Gil Privé who offered invaluable guidance, assistance and patience. Special thanks are also due to the members of my supervisory committee, Dr. Avi Chakrabarty and Dr. Mitsu Ikura. Without their knowledge and assistance this study would not have been successful. Deepest gratitude to all past and present members of the Privé lab and the Rose lab who provided a positive atmosphere for knowledge exchange, I learned so much from all these great scientists. Especially Peter Stogios, Tine Nielsen, Alex Ghetu, John Holyoake, Wesley Errington, Dona Ho and Konstatin Popovic for their helpful discussions; Lu Chen and Neil Pomroy for technical supports; as well as Eden Fussner and Seiji Sugiman-Marangos for their input to this project. I would also like to convey my thanks to all labs at 4th floor of TMDT for instrument usages. Last but not least, I want to thank my beloved families in China and all my friends in Canada for their understanding and endless love throughout these two years. iii Contents Abstract .................................................................................................................................. ii Acknowledgements ............................................................................................................ iii Contents ................................................................................................................................ iv List of tables ........................................................................................................................ vii List of figures ..................................................................................................................... viii List of equations ................................................................................................................. ix List of abbreviations ........................................................................................................... x Chapter 1: Introduction ............................................................................................... 1 1.1 Overview of BTB domains ............................................................................................ 1 1.2 BTB-Zinc Finger proteins ............................................................................................ 1 1.2.1 BTB-Zinc Finger proteins as transcriptional regulators................................................. 1 1.2.2 Proteins of interest .............................................................................................................. 3 1.3 BTB domain-domain interactions ............................................................................... 5 1.3.1 Transcription factors interactions ..................................................................................... 5 1.3.2 BTB domain homodimers: functional and structural highlights ................................... 8 1.3.3 BTB domain heterodimers: findings and biological roles .............................................. 11 1.4 Biological roles of 14 BTB domains ........................................................................... 13 PLZF ........................................................................................................................................13 BCL6 ........................................................................................................................................15 MIZ1 ........................................................................................................................................16 RP58 .........................................................................................................................................17 BOZF .......................................................................................................................................18 Y441 .........................................................................................................................................19 HKR3 .......................................................................................................................................19 Kaiso ........................................................................................................................................19 iv ZID ...........................................................................................................................................20 FAZF ........................................................................................................................................21 Bioref ........................................................................................................................................22 KUP ..........................................................................................................................................22 ZBTB4 ......................................................................................................................................22 LRF ..........................................................................................................................................23 1.5 Project goals ................................................................................................................. 25 1.6 Backgrounds in assay development ........................................................................... 25 1.6.1 Overview of protein-protein interaction assays ..............................................................25 1.6.2 Fluorescent protein tags ....................................................................................................28 1.6.3 Kinetics in BTB dimer formation .....................................................................................29 1.6.4 Chemical denaturation and refolding ..............................................................................31 1.6.5 The coexpression system ...................................................................................................31 Chapter 2: Materials and Methods .............................................................................. 33 2.1 Fluorescent protein labeling and optimization ......................................................... 33 2.1.1 Fluorescent protein background minimizations .............................................................33 2.1.2 Placement of fluorescent proteins to BTB domains ........................................................35 2.2 Dimer formation by chemical denaturation and refolding ..................................... 35 2.2.1 Protein expression and purification .................................................................................35 2.2.2 Kinetic studies of BTB domain interactions ....................................................................36 2.2.3 Dimer formation by chemical denaturation and renaturation ......................................37 2.3 Dimer formation by co-expression system ................................................................ 39 2.3.1 Cloning: insertion of BTB domains into co-expression vectors .....................................39 2.3.2 BTB pairs co-expression in auto-induction media ..........................................................40 2.3.3 Detection of dimer formation ...........................................................................................42 2.3.4 Data analysis ......................................................................................................................43 Chapter 3: Results ............................................................................................................. 44 3.1 Fluorescent protein interactions ................................................................................ 44 3.2 Results from chemical denaturation and refolding assay .......................................