Abstract
The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.
Highlights
Gene expression is precisely regulated by a combination of promoters and gene-distal regulatory regions, known as enhancers[1,2]
This review summarizes the assays developed for functional genomewide testing of enhancer activity and their limitations as well as the main findings that have been gathered using these techniques
We developed a capture-based approach to assess a subset of mouse DNase I hypersensitive sites (DHSs) found in developing thymocytes[22]
Summary
Gene expression is precisely regulated by a combination of promoters and gene-distal regulatory regions, known as enhancers[1,2]. A current limitation of these approaches is that the screening strategy might be based on phenotypic features (such as cell growth fitness, developmental markers, etc.) instead of directly assessing the expression levels of regulated genes To overcome this limitation, a new powerful method combined CRISPRi and single-cell RNA-seq[48], enabling high-throughput interrogation of enhancers at single-cell resolution and directly linking enhancer function, and their combinations, with its target gene(s). A new powerful method combined CRISPRi and single-cell RNA-seq[48], enabling high-throughput interrogation of enhancers at single-cell resolution and directly linking enhancer function, and their combinations, with its target gene(s) These approaches have been used so far to scan restricted genomic areas, they will likely be implemented in true genome-wide screens of regulatory elements in the coming future. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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