The molecular role of GTF2IRD1: a protein involved in the neurodevelopmental abnormalities of Williams-Beuren syndrome Paulina Carmona-Mora A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical Sciences, Faculty of Medicine UNSW Australia Sydney, Australia May 2015 i ii ACKNOWLEDGMENTS I would like to express my gratitude to the people that have been with me in this important stage of my life. First, infinite thanks to my supervisors, Prof. Edna Hardeman and Dr Stephen Palmer, for your continual support throughout these years and for mentoring me. My PhD has been an intense and challenging journey, both at the professional and personal level, but I am thankful I went through this road with you. Both were always there for understanding and encouraging me, but also to enjoy the rewarding results of this experience. I will definitely take with me what I learned from you, Edna; I admire you as a strong, experienced and successful woman in science. Steve, your attitude so sensible and patient has shaped the way I want to do science. The Cellular and Genetic Medicine Unit is an enriching and multicultural environment that made my time here so enjoyable. Thanks to Prof. Peter Gunning, your lively interest in science is pure inspiration. I also want to thank the CGMU supervisors Dr Galina Schevzov and Dr Annemiek Beverdam for their advice and delightful conversations. I am grateful for having great people around me in the lab, first of all Florence Tomasetig, who besides being so important in the project, helped me with the experiments and made our days in the lab so vibrant and pleasant, your friendship is a real gem. I also want to thank you April, Wei, Cecilia, Jeff and Annamaria for being so helpful and cheerful. To my fellow PhD students, Bin, Melissa, Iman, Bassem, Veronica and Nadia, we shared the bitter, the sweet and the celebrations of this experience. I was fortunate to collaborate with great scientists from whom I learned so much. Thanks to Prof. Mark Wilkins and Natalie Twine, from the NSW Systems Biology Initiative, for their help with the microarray experiments. Thanks to Iveta Slapetova and Dr Renee Whan, from the Biomedical Imaging Facility for their excellent training and assistance with the confocal microscopy. And thanks to Dr Mirella Dottori, from the University of Melbourne for sharing her expertise on embryonic stem cells and provide us with human neurospheres. I dedicate my achievements to Cesar, my husband and lab mate, indeed this PhD candidature has been a shared effort, both in the lab and at home; without your understanding and love I would not have been able to finish this work. To my family in Chile, my mom, dad and sisters Emma and Valeria, for their endless support and source iii of strength. To my son, Diego, who without knowing, is my constant motivation for organising and balancing my life and who is my inspiration to go through everything life brings. iv PUBLICATIONS ARISING FROM WORK PRESENTED IN THIS THESIS P. Carmona-Mora, J. Widagdo, F. Tomasetig, CP. Canales, Y. Cha, WS. Lee, A. Alshawaf, M. Dottori, EC. Hardeman, SJ. Palmer (2015) The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation. (Submitted to Human Genetics, in production of revised manuscript). In preparation: P. Carmona-Mora, F. Tomasetig, CP. Canales, A. Alshawaf, M. Dottori, JI. Young, L. Hesson, R. Barres, EC. Hardeman, SJ. Palmer (2015) The epigenetic role of GTF2IRD1 as a means to understand features of Williams-Beuren syndrome. Selected conference abstracts P. Carmona-Mora, J. Widagdo, KM. Taylor, R. Tsz-Wai Pang, PW. Gunning, EC. Hardeman, SJ. Palmer (2012) The molecular role of GTF2IRD1, a protein implicated in the neurodevelopmental features of Williams-Beuren syndrome. 62th Annual Meeting, The American Society of Human Genetics. San Francisco, CA, USA. P. Carmona-Mora, J. Widagdo, KM. Taylor, R. Tsz-Wai Pang, F. Tomasetig, NA. Twine, MR. Wilkins, PW. Gunning, EC. Hardeman, SJ. Palmer (2013) The molecular role of the transcriptional regulator GTF2IR1 in the pathogenesis of Williams-Beuren syndrome. 34th Lorne Genome Conference, Lorne, Vic, Australia. P. Carmona-Mora, J. Widagdo, F. Tomasetig, KM. Taylor, R. Tsz-Wai Pang, NA. Twine, MR. Wilkins, PW. Gunning, EC. Hardeman, SJ. Palmer (2013) A gene v implicated in the neurobehavioural abnormalities of Williams-Beuren syndrome, GTF2IRD1, is a novel epigenetic regulator. Genetics Society of Australasia, Sydney, NSW, Australia. P. Carmona-Mora, J. Widagdo, F. Tomasetig, KM. Taylor, Y. Cha, R. Tsz-Wai Pang, NA. Twine, MR. Wilkins, PW. Gunning, EC. Hardeman, SJ. Palmer. A gene implicated in the neurobehavioural abnormalities of Williams-Beuren syndrome, GTF2IRD1, encodes a novel epigenetic regulator (2013) ComBio, Perth, WA, Australia. (Oral presentation) P. Carmona-Mora, J. Widagdo, F. Tomasetig, KM. Taylor, Y. Cha, R. Tsz-Wai Pang, NA. Twine, MR. Wilkins, PW. Gunning, EC. Hardeman, SJ. Palmer (2013) A gene implicated in the neurobehavioural abnormalities of Williams-Beuren syndrome, GTF2IRD1, encodes a novel epigenetic regulator. 63th Annual Meeting The American Society of Human Genetics, Boston, MA, USA. (Oral presentation) P. Carmona-Mora, F. Tomasetig, CP. Canales, A. Alshawaf, M. Dottori, EC. Hardeman, SJ. Palmer (2014) Unravelling epigenetic complexes associated with GTF2IRD1 to understand the cognitive features of Williams-Beuren syndrome. ComBio, Canberra, ACT, Australia. (Oral presentation) P. Carmona-Mora, F. Tomasetig, CP. Canales, A. Alshawaf, M. Dottori, EC. Hardeman, SJ. Palmer (2014) Defining the presence of GTF2IRD1 in epigenetic complexes as a means to understand features of Williams-Beuren syndrome. 64th Annual Meeting The American Society of Human Genetics, San Diego, CA, USA. vi ABSTRACT Background: GTF2IRD1 is a member of the GTF2I gene family, located on chromosome 7 in a region prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. Human mapping data and analyses of mouse knockouts implicate GTF2IRD1 as the prime candidate for the craniofacial abnormalities, mental retardation, visuospatial construction deficits and hypersociability of WBS. Aims: The aim of this work was to study the cellular and molecular role of GTF2IRD1 by investigating: i) the cellular localisation of GTF2IRD1; ii) its protein interacting partners; iii) the gene dysregulation caused by GTF2IRD1 loss; and iv) the presence of GTF2IRD1 in epigenetic complexes regulating gene expression. Results: i) Immunofluorescence analyses in mammalian cell lines and in human ES cell-derived neurons showed endogenous GTF2IRD1 as a nuclear speckle protein. The comparison of this punctate pattern with markers of nuclear sub-compartments and chromatin marks supports an association with developmentally regulated silent chromatin. ii) To define functional relationships, yeast two-hybrid screenings were used to isolate novel interaction partners. Most of the nuclear-localised interactions were validated in mammalian cells, being predominantly proteins involved in chromatin modification and transcriptional regulation. The sites of interaction in GTF2IRD1 were mapped to specific domains. iii) To identify transcriptional changes arising from GTF2IRD1 loss, microarray studies were conducted in siRNA-treated HeLa cells and brain tissue from Gtf2ird1 knockout mice. In the corpus striatum, qPCR validation indicated up-regulation of genes involved in neuronal development and immediate-early response genes that may explain some of the observed neurobehavioural phenotypes. iv) vii GTF2IRD1 was found to be involved in chromatin modifying complexes by direct associations with histone deacetylases and can affect their enzymatic activity. Conclusions: The results of this thesis indicate that GTF2IRD1 forms complexes with DNA-binding and chromatin modifying proteins to regulate gene expression through epigenetic mechanisms that are controlled in a tissue specific manner. The sites of protein interactions indicate key features regarding the evolution of GTF2IRD1 and integration with tight post-translational regulation, fitting well with the concept of human disease states caused by copy number variation. viii ABBREVIATIONS Ade adenine ADHD attention deficit hyperactive disorder ASD autism spectrum disorder BCA bicinchoninic acid assay BSA bovine serum albumin ChIP chromatin immunoprecipitation Co-IP coimmunoprecipitation CNV copy number variation DDO double dropout DMEM Dulbecco’s modified Eagle’s medium DNA deoxyribonucleic acid DNAse deoxyribonuclease ECL enhanced chemiluminescence GFP green fluorescent protein GUR GTF2IRD1 upstream region HDAC histone deacetylase His Histidine IgG immunoglobulin G IP immunoprecipitation LCR low copy repeat Leu leucine Lys lysine LZ leucine zipper NAHR non-allelic homologous recombination NLS nuclear localisation signal OMIM online mendelian inheritance in man ORF open reading frame PBS phosphate-buffered saline PCR polymerase chain reaction ix PFA paraformaldehyde PLA proximity ligation assay QDO quadruple dropout qRT-PCR quantitative real time PCR RD repeat domain RNA ribonucleic acid SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM standard error of the mean SUMO small ubiquitin-like modifier SVAS supravalvular aortic stenosis TAE tris-acetate-EDTA buffer Trp tryptophan UV ultra violet WBS Williams-Beuren syndrome WSCP Williams syndrome cognitive profile WBSCR