Massively parallel reporter assays: from barcodes to biology

Subjects

Unlike well-defined triplet codons or splicing sites of protein-coding genes, cis-regulatory elements such as enhancers and promoters lack a strict sequence code, making them difficult to identify across the vast non-coding regions of the genome. Since the 2000s, researchers in functional genomics have successfully identified these elements by leveraging evolutionary conservation and cell-type-specific biochemical features, such as histone modifications or chromatin accessibility. However, profiling approaches such as ATAC-seq, ChIP–seq or DNase-seq remain fundamentally descriptive; they capture the endogenous genomic or epigenomic landscape of cells without directly probing regulatory function. Answering the long-standing question of which non-coding variants give rise to disease phenotypes or evolutionary change has therefore relied heavily on low-throughput experimental strategies, including mouse genetics and conventional luciferase or lacZ reporter assays.

Another considerable advance achieved by this work was the application of saturation mutagenesis to the systematic analysis of transcriptional regulation. The authors focused on a bacteriophage promoter and mammalian core promoters, generating comprehensive libraries that incorporated all possible nucleotide substitutions and deletions within defined promoter regions. For example, they synthesized a CMV promoter sequence containing nucleotide substitutions or deletions at each nucleotide position spanning −45 bp to +25 bp relative to the transcription start site, making a total of 280 variants. Each variant was associated with multiple unique DNA barcodes, thereby increasing measurement robustness. Regulatory activity was quantified by assessing barcode transcription levels in an in vitro transcription system or in mammalian cells. This strategy enabled large-scale functional dissection of promoter activity at single-nucleotide resolution and, notably, led to the experimental rediscovery of the TATA box as a key determinant of transcriptional activity — as originally identified in the 1970s by Goldberg and Hogness on the basis of sequence conservation. A follow-up study by Patwardhan et al. in 2012 further extended this strategy by analysing distal enhancers in the mouse liver in vivo, demonstrating the broader applicability of MPRAs to more complex regulatory contexts.

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