Abstract
Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.
Highlights
Mutations or single-nucleotide polymorphisms (SNPs) in noncoding regions of the human genome functioning as cis-regulatory elements (CREs) or enhancers have been widely implicated in human disease and disease predisposition (Bhatia and Kleinjan, 2014; Chatterjee and Ahituv, 2017)
The widespread application of whole-genome sequencing for understanding genetic diseases, combined with genome- wide identification of chromatin signatures associated with active enhancers, has led to the identification of a large number of putative enhancers with disease-a ssociated or disease risk-a ssociated sequence variation (Bhatia and Kleinjan, 2014; Chatterjee and Ahituv, 2017; Short et al, 2018; Wu and Pan, 2018)
CRE activity depends on precise stoichiometric concentrations of specific
Summary
Mutations or single-nucleotide polymorphisms (SNPs) in noncoding regions of the human genome functioning as cis-regulatory elements (CREs) or enhancers have been widely implicated in human disease and disease predisposition (Bhatia and Kleinjan, 2014; Chatterjee and Ahituv, 2017). Enhancer-reporter transgenic assays have been widely employed in a variety of model organisms, including the mouse, to assess enhancer function in vivo (Ashery-Padan and Gruss, 2001; Bhatia et al, 2015; Farley et al, 2015; Rogers and Williams, 2011; Visel et al, 2007). These assays can be affected by the random integration of transgenes and have
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