Functional analysis of human enhancers using the zebrafish embryo

[摘要] 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.

[发布机构] University:University of Birmingham;Department:School of Clinical and Experimental Medicine

[关键词] Q Science;QH Natural history;QH426 Genetics