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Function of Sox2 As a Transcriptional Repressor

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Function of Sox2 As a Transcriptional Repressor Function of Sox2 as a transcriptional repressor By Yu-Ru Liu, M. Sc Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy September 2011 1 Abstract Sox2 is one of the earliest known transcription factors to be expressed during development of the nervous system (Rex et al., 1997; Silvia Brunelli, 2003; Wang et al., 2006b; Dee et al., 2008). Ectodermal cells expressing Sox2 have the potential to differentiate into nerve cells. Cells expressing Sox2 are specified to a neural fate during neural induction. Sox2 belongs to the SoxB1 family, comprising Sox1, Sox2 and Sox3, which are generally considered to activate specific target genes, whereas, the SoxB2 group, Sox14 and Sox21, act as transcriptional repressors (Uchikawa, Kamachi, & Kondoh, 1999). However, Sox2 has also been demonstrated to act as a repressor (Kopp et al., 2008) which implies that Sox2 could have a dual-function in vivo . Previous studies indicated that the HMG box-containing protein, Tcf/Lef, interacts with the transcriptional co-repressor, Groucho (Helen Brantjes, 2001). We therefore set out to determine if interaction with the Groucho co-repressor could also explain the repressor ability of Sox2. In this study, we have examined the interaction between Sox2 and Groucho using nuclear translocation, yeast-two-hybrid and co-immunoprecipitation assays. The data suggest that Sox2 interacts with Groucho through a C-terminal, engrailed-like motif. The effect of Groucho on Sox2 function was measured using a luciferase reporter assay. The transcriptional activation activity of Sox2 was repressed after co-expressing with Groucho. To address the biological function of Sox2-Groucho interaction, a loss-of-repressor-function mutant of Sox2 was created by point mutating the essential engrailed-like motif. Analysis in Zebrafish embryos indicated that the of loss-of-repressor-function mutant of Sox2 (Sox2 LQY/AAA ) lost the ability to repress the expression of chordin . In 1 human neural stem cells, Affymetrix arrays revealed that 676 genes were activated and 786 genes were repressed by Sox2 overexpression. Within the genes that were repressed by Sox2, approximatly 7% were less repressed by Sox2 LQY/AAA . Together, these data suggest that Sox2 functions not only as a transcriptional activator but also as a repressor through interacting with the co- repressor Groucho. However, because only 7% of the repressed genes were affected by the Sox2 LQY/AAA mutant, this suggests that there are other mechanisms involved in Sox2 transcriptional repressor function. 2 Publications Yu-Huan Shih, Cheng-Liang Kuo, Caroline S. Hirst, Chris T. Dee, Yu-Ru Liu , Zulfiqar Ali Laghari and Paul J. Scotting (2010). SoxB1 transcription factors restrict organizer gene expression by repressing multiple events downstream of Wnt signalling. Development . 137, 2671-2681. 3 Acknowledgments First of all, I would like to thank my supervisor Dr. Paul J. Scotting for giving me the opportunity to work in his lab and for his valuable guidance and optimistic attitude throughout my studies. Thanks also go to Professor Jane Hewitt for her useful suggestions and encouragement. Big thank-you goes to Zulfiquar for his endless support and friendship. To Dr. Jennie N. Jeyapalan, thanks for her time in patiently listening and encouraging me to pursue a career in science. I would also like to thank Ivan, Peggy, Sarah and Thomas for soothing me from homesickness. Thanks to Elaine and Magda for their technical support. To Chris Tan, Chris Dee and Dylan for the interesting scientific chats. Caz, Dzul, Michelle and all the lab members, thanks for sharing enthusiasm and team spirit. Caro, Tom, Paul Goodwin and all the master students worked with me, thanks for your help and friendship. A special thank goes to Remy and my families for balancing my life outside work. Belinda, Yi-Fang, Zora and all my friends, thanks for the accompaniment and support. I would like to dedicate this thesis to my mother in heaven. I could not fulfil my dream without her. 4 List of Contents Chapter 1 Introduction .................................................................................... 1 1.1 SoxB1 in early neural development ...................................................... 1 1.1.1 Neural induction ....................................................................................... 1 1.1.2 Transcription factors ................................................................................. 5 1.1.3 Sox family .................................................................................................. 6 1.1.4 SoxB1 subgroup ........................................................................................ 8 1.1.5 Sox B1 genes in neural development ..................................................... 10 1.2 Sox2 in stem cells ............................................................................... 14 1.2.1 Stem cells ................................................................................................ 14 1.2.2 Sox factors in neural stem cells .............................................................. 15 1.2.3 Sox2 in the core transcriptional network of the ES cell .......................... 18 1.2.4 Sox2 in induced pluripotent stem (ips) cells ........................................... 20 1.3 Sox2 as a transcription factor ............................................................. 22 1.3.1 The transcriptional activation activity domain of the SoxB1 subgroup .. 22 1.3.2 Partners of SoxB1 transcriptional activation activity .............................. 25 1.3.3 Sox B1 genes as transcriptional repressors ............................................ 27 1.4 Co-repressors of transcription ............................................................ 29 1.4.1 Groucho .................................................................................................. 30 1.4.2 Groucho interaction motifs in transcription factors ............................... 33 1.4.3 Long form and short form of Groucho ................................................... 36 1.4.5 Functions of Groucho in development ................................................... 37 1.4.6 Groucho and HMG transcription factors ................................................ 38 1.4 Research objectives ............................................................................ 38 Chapter 2 Materials and methods ................................................................. 40 2.1 Construction of Sox2 mutants expression plasmid ............................. 40 2.1.2 Measuring DNA concentrations .............................................................. 40 2.1.3 Nucleic acid restriction digests ............................................................... 40 2.1.4 Agarose gel electrophoresis ................................................................... 41 2.1.5 DNA purification from agarose gel ......................................................... 41 2.1.6 DNA ligation and bacterial transformation............................................. 41 2.1.7 Glycerol stock of bacteria ....................................................................... 42 2.1.8 Polymerase chain reaction (PCR) ............................................................ 42 2.1.9 TA cloning ............................................................................................... 44 5 2.1.10 Site directed mutagenesis .................................................................... 44 2.1.11 DNA sequencing .................................................................................... 45 2.2 Cell culture ......................................................................................... 46 2.2.1 COS-7 and P19 cell maintenance ............................................................ 46 2.2.2 hNSC cell maintenance ........................................................................... 46 2.2.3 Storage and revival of cells in frozen stocks ........................................... 46 2.2.4 Cell transfection by electroporation ....................................................... 47 2.2.5 Cell transfection by liposome reagent .................................................... 48 2.3 Protein analysis ................................................................................... 49 2.3.1 Cell preparation and Co-immunoprecipitation ....................................... 49 2.3.2 Crosslinker treated cell lysate preparation ............................................. 49 2.3.3 SDS-PAGE electrophoresis ...................................................................... 50 2.3.4 Western blot ........................................................................................... 51 2.3.5 Cell preparation and immunostaining .................................................... 52 2.3.6 Cell preparation and Luciferase assay .................................................... 52 2.4 Yeast manipulations ........................................................................... 54 2.4.1 Yeast maintenance ................................................................................. 54 2.4.2 Preparation of yeast competent cells ..................................................... 54 2.4.3 Lithium acetate mediated yeast transformation .................................... 54 2.4.4 Protein detection in transformed yeast ................................................. 55 2.4.5 Detection of protein interaction by yeast-two-hybridization ................ 55 2.5 hNSC mRNA manipulation
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