Recent advances in high-throughput approaches to dissect enhancer function

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.

Keywords: CRISPR; MPRA; STARR-seq; enhancer function; gene transcription.

Figure 1.. Principle of high-throughput assays for enhancer activity.

( A) Overview of massively parallel reporter assay (MPRA). The test sequences (wild-type, variants, etc.) are generally synthesized in silico by massive oligonucleotide synthesis with unique barcode tags and cloned into the plasmid backbone. Tags can be synthesized along with the test sequences or added after synthesis by polymerase chain reaction (PCR) amplification. A basal promoter and a reporter open reading frame (ORF) are inserted between the tested element and tag sequences. The reporter library is then transfected into cultured cells. Subsequently, mRNA is isolated and cDNA synthesized. The tags are sequenced before (plasmid library pool, for normalization) and after the transfection. The difference in the enrichment of each barcode is proportional to the enhancer activity of the test sequence. In the case of post-synthesis addition of barcodes, an additional sequencing step is required at the first cloning step. ( B) Overview of self-transcribing active regulatory region sequencing (STARR-Seq). A genomic or bacterial artificial chromosome (BAC) library is cloned in the reporter plasmid, downstream of the ORF and upstream of the polyadenylation site (pAS). Alternatively, the regions of interest might be enriched by a capture approach. The reporter library is transfected into cultured cells. Subsequently, mRNA is isolated and cDNA synthesized. The cloned regions are sequenced from the plasmid library pool (input) and the cDNA. Differences in the enrichment with respect to the input are proportional to the enhancer activity. In both panels, the effect of the enhancer on the basal promoter is indicated by an arrow.