Decoding enhancers using massively parallel reporter assays

Abstract

Enhancers control the timing, location and expression levels of their target genes. Nucleotide variation in enhancers has been shown to lead to numerous phenotypes, including human disease. While putative enhancer sequences and nucleotide variation within them can now be detected in a rapid manner using various genomic technologies, the understanding of the functional consequences of these variants still remains largely unknown. Massively parallel reporter assays (MPRAs) can overcome this hurdle by providing the ability to test thousands of sequences and nucleotide variants within them for enhancer activity en masse. Here, we describe this technology and specifically focus on how it is being used to obtain an increased understanding of enhancer regulatory code and grammar.

Keywords: Enhancer; Massively parallel reporter assays; Transcriptional regulation.

Fig. 1

Comparison of plasmid construct, assay model, strategy of reporter detection and advantage and disadvantages of different MPRA technologies [, –36, 38, 39]. ARS-CEN, yeast autonomously replicating sequence and centromere; BC, barcode; HygroR, hygromycin-resistance gene; LTR, long terminal repeat; P, promoter; pA, polyadenylation signal; TR, terminal repeat, Ub, ubiquitin promoter.

Fig. 2

Overview of the massively parallel reporter assay. (A) To generate a short enhancer MPRA library, candidate enhancer sequences and barcode sequences are synthesized on a programmable microarray and cloned into a plasmid vector. Minimal promoter (mP) and EGFP reporter gene are inserted between the enhancer and barcode. (B) Polymerase cycling assembly is used to generate a long enhancer library. Long enhancer variants (red vertical lines) and barcodes are separately inserted upstream of mP or within the 3’ UTR of EGFP gene, respectively. (C) To associate enhancer and barcodes, the downstream end of the enhancer and upstream end of the barcode are digested and re-ligated to bring them adjacent to one another, followed by sequencing with tag-guided subassembly. (D) The MPRA library is introduced into cell lines or tissues. Transcribed barcode RNAs are extracted from the cells. (E) The relative transcriptional activities of distinct enhancers are measured by sequencing and counting their corresponding barcode RNAs. The counts of transcribed RNA barcodes are normalized by DNA barcode counts that are from the library. BC, barcode; mP, minimal promoter; pA, polyadenylation signal.