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Global Analysis of Gene Expression in the Developing Brain of Gtf2ird1

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Global Analysis of Gene Expression in the Developing Brain of Gtf2ird1 Global Analysis of Gene Expression in the -/- Developing Brain of Gtf2ird1 Mice by Jennifer Anne O’Leary A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Jennifer Anne O’Leary (2011) Global Analysis of Gene Expression in the Developing Brain of -/- Gtf2ird1 Mice Jennifer Anne O’Leary Doctor of Philosophy Department of Molecular Genetics University of Toronto 2011 Abstract Williams-Beuren Syndrome (WBS) is an autosomal dominant neurodevelopmental disorder caused by hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23. Symptoms are numerous and include behavioural and cognitive components. One of the deleted genes, GTF2IRD1, a putative transcription factor, has been implicated in the neurological features of WBS by studying patients with atypical deletions of 7q11.23. Gtf2ird1-targeted mice have features consistent with the WBS phenotype, namely reduced innate fear and increased sociability. To identify neural targets of GTF2IRD1, microarray analyses were performed comparing gene expression in whole brains of Gtf2ird1-/- and wildtype (WT) mice at embryonic day 15.5 and at birth. Overall, the changes in gene expression in the mutant mice were not striking, with most falling in the range of 0.3 to 2 fold. qRT-PCR was used to verify the expression levels of candidate genes and examination of verified genes revealed that most were located on chromosome 5, within 50 Mb of Gtf2ird1. Expression of these candidate genes in -/- Gtf2ird1 mice was found to be the same as in WT 129S1/SvImJ mice, indicating the ii differences were the result of flanking chromosomal material from the, 129-derived, R1 ES cells from which the Gtf2ird1-/- mice were generated, and that expression differences were unrelated to Gtf2ird1 dosage. Further analysis found that while many genes showed decreased expression using primers targeting the 3’ UTR, expression of upstream exons was not affected. Transcripts using alternative polyadenylation sites were identified using 3’ RACE, and qRT-PCR showed that expression of different 3’ UTR isoforms can occur in a strain specific manner. Expression analysis of previously identified GTF2IRD1 targets also failed to demonstrate an in vivo effect. In summary, I was unable to find any in vivo neuronal targets of this putative transcription factor, despite its robust expression in the developing rodent brain. iii Acknowledgements The work that I completed over the past seven years would not have been possible without the help and support of many people. First, I must thank my supervisor, Dr. Lucy Osborne for her guidance and support. It has been a pleasure working in her lab, and her ability to put a positive spin on my negative results kept me from getting too depressed as the list of genes that Gtf2ird1 does not regulate continued to grow. I would also like to thank my supervisory committee members, Dr. Sabine Cordes and Dr. Timothy Hughes for their helpful insights, suggestions and technical assistance. All members of the Osborne lab, past and present, have made the lab a great environment to work in. I will greatly miss the countless hours of “scientific” discussion, cookie days, and their company during “coffee time”. In particular, I owe a big thank you to Ted Young for teaching me how to be a good scientist. Although his Lil John impressions drove me crazy, he always came through with helpful advice when it was most needed. Finally, none of this would have been possible without the continued support of my family, especially my parents. I was the first of their four children to enter university, and the last one to leave. Their unconditional love and encouragement have undoubtedly made it possible for me to be where I am today. iv Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iv Table of Contents .......................................................................................................................... v LIST OF TABLES ........................................................................................................................ x LIST OF FIGURES ..................................................................................................................... xi LIST OF ABBREVIATIONS ................................................................................................... xiii Chapter I: Introduction ................................................................................................................ 1 1.1 Williams-Beuren syndrome ................................................................................................ 1 1.1.1 History of Williams-Beuren syndrome ....................................................................... 1 1.1.2 Williams-Beuren syndrome clinical phenotype .......................................................... 2 1.1.3 The Williams-Beuren syndrome cognitive phenotype ............................................... 5 1.1.4 The Williams-Beuren syndrome behavioural phenotype ........................................... 8 1.2 The genetic basis of Williams-Beuren syndrome ............................................................... 9 1.2.1 Identification of a microdeletion at 7q11.23 ............................................................... 9 1.2.2 Genomic rearrangements at 7q11.23 ........................................................................ 12 1.2.3 Atypical deletions in the Williams-Beuren syndrome region ................................... 15 1.3 General Transcription Factor 2-I (GTF2-I) gene family ................................................... 17 1.3.1 General Transcription Factor 2-I (TFII-I) ................................................................. 18 1.3.2 General Transcription Factor 2-I Repeat Domain containing 1 (TFII-IRD1) .......... 21 1.3.3 General Transcription Factor 2-I Repeat Domain containing 2 (TFII-IRD2) .......... 24 v 1.4 The Gtf2ird1 mouse model ............................................................................................... 25 1.4.1 Generation of the mouse model ................................................................................ 26 1.4.2 Behavioural phenotypic analysis .............................................................................. 28 1.4.3 Biochemical and electrophysiological phenotypic analysis ..................................... 29 1.5 Research Aims and Hypothesis ........................................................................................ 29 Chapter II: TFII-IRD1 may not function as a transcription factor in the developing mouse brain. ............................................................................................................................................ 31 2.1 Abstract ............................................................................................................................. 31 2.2 Literature Review .............................................................................................................. 32 2.2.1 Evidence supporting the role of TFII-IRD1 as a transcription factor ....................... 32 2.2.2 Cellular localization of TFII-IRD1 ........................................................................... 39 2.3 Material and Methods ....................................................................................................... 41 2.3.1 Generation of probes for in situ hybridization .......................................................... 41 2.3.2 Whole mount in situ hybridization of Gtf2ird1-/- embryos ....................................... 42 2.3.3 In situ hybridization of P0 mouse brain sections ...................................................... 44 2.3.4 Preparation and culture of mouse embryonic fibroblast (MEF) cells ....................... 45 2.3.5 Dissection of mouse tissues and RNA isolation ....................................................... 46 2.3.6 Genotyping of P0 and embryonic mice .................................................................... 46 2.3.7 Microarray analysis using the Affymetrix mouse 430 2.0 gene chip ....................... 47 2.3.8 Microarray analysis using the Illumina mouseWG-6 v2.0 BeadChip ...................... 48 2.3.9 Expression analysis using quantitative Real-Time PCR ........................................... 49 2.3.10 siRNA knockdown of Gtf2ird1 in neuronal cell lines .......................................... 52 2.3.11 Cellular localization of Gtf2ird1 in Neuro2a cells ................................................ 53 2.3.12 Expression analysis using western blots ............................................................... 55 vi 2.4 Results ............................................................................................................................... 56 2.4.1 Gtf2ird1 is expressed in the developing mouse brain ............................................... 56 2.4.2 Expression of candidate target genes Hoxc8 and Gsc are not altered in E11.5 Gtf2ird1-/- mouse embryos .................................................................................................... 56 2.4.3 Expression of TFII-IRD1 candidate target genes identified in vitro are not altered in vivo 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