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Functional analysis of human enhancers using the zebrafish embryo

Miguel Escalada, Irene (2014)
Ph.D. thesis, University of Birmingham.

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Abstract

In the post-genomic era the availability of genome-wide datasets has revealed an unexpected complexity of transcriptional regulation. In this context, where most enhancer predictions are based on computational analyses, functional validations are lacking. This thesis investigated the utility of the transgenic zebrafish embryo as an in vivo vertebrate model to study the function of candidate human enhancers, and detect subtle changes in enhancer function caused by disease-associated variants. Our functional validations indicated that despite the evolutionary distance between human and fish, 60% of the conserved enhancers predicted by a combination of chromatin signatures, TF binding events and bidirectional transcription, lead to reporter expression that recapitulates the patterns of either zebrafish or human genes. To improve the reliability of zebrafish transgenesis, a targeted integration system mediated by PhiC31 integrase was validated for enhancer testing. I demonstrated that this method overcomes position effect variation commonly found in transposon-based assays. However, enhancer-driven expression could not be detected when I attempted to quantitate TCF7L2-associated enhancer variants, indicating the need for further studies to understand the limitations of the zebrafish model. Taken together, my results provide strong support for zebrafish as a valuable in vivo model to study the function of mammalian transcriptional regulatory elements.

Type of Work:Ph.D. thesis.
Supervisor(s):Muller, Ferenc and Gissen, Paul
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Clinical and Experimental Medicine
Subjects:QH426 Genetics
QR Microbiology
Institution:University of Birmingham
ID Code:5268
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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