Abstract
Determining aetiology of genetic disorders caused by damaging mutations in protein-coding genes is well established. However, understanding how mutations in the vast stretches of the noncoding genome contribute to genetic abnormalities remains a huge challenge. Cis-regulatory elements (CREs) or enhancers are an important class of noncoding elements. CREs function as the primary determinants of precise spatial and temporal regulation of their target genes during development by serving as docking sites for tissue-specific transcription factors. Although a large number of potential disease-associated CRE mutations are being identified in patients, lack of robust methods for mechanistically linking these mutations to disease phenotype is currently hampering the understanding of their roles in disease aetiology. Here, we have described the various systems available for testing the CRE potential of stretches of noncoding regions harbouring mutations implicated in human disease. We highlight advances in the field leading to the establishment of zebrafish as a powerful system for robust and cost-effective functional assays of CRE activity, enabling rapid identification of causal variants in regulatory regions and the validation of their role in disruption of appropriate gene expression.
Highlights
Human genetic diseases affect a wide range of tissues and are caused by numerous types of mutations
It has been firmly established that many of these mutations reside in the noncoding regions of the human genome, most of which are likely to harbour cis-regulatory elements (CREs) [1,2]
CRE sequences are highly enriched for binding sites of tissue-specific transcription factors (TF)
Summary
Human genetic diseases affect a wide range of tissues and are caused by numerous types of mutations. Advances in genomic sequencing technologies have enabled widespread application of whole genome sequencing technology to patient DNA samples These studies have led to the identification of clinically-relevant mutations in the noncoding regions of the human genome, most of which are likely disrupting CRE function of these sequences by altering the sequences of transcription factor binding sites [4]. Chromatin looping studies by a variety of chromosome conformation capture techniques (e.g., 3C, 4C, 5C, Hi-C, ChIA-PET), DAM-ID and 3D FISH [14,15] have been employed to detect interactions between predicted CREs and their target gene promoter, in the context of the entire gene locus These assays have been performed on a large number of in vitro cultured cell lines, whole embryos and tissues derived from human, mouse and zebrafish [16]. Recent advances in single-cell technologies [11] have the potential for overcoming this limitation, provided robust methods of obtaining the precise cell-types where the CREs are active are developed
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