UC San Diego

Transcription is the fundamental mode of action by which the genome carries out its function. Where and when transcription occurs is precisely controlled by regulatory elements, such as promoters and enhancers. While many sequencing techniques have been established that have allowed the identification and annotation of these regulatory elements, the proper understanding how these regulatory sequences encode their function remains difficult to parse, largely due to issues in carrying out the massive scale of sequence perturbations required to properly understand sequence-function encoding in vivo. Massively parallel reporter assays (MPRAs) overcome this limitation by enabling the functional screening of thousands of regulatory sequences en masse. We developed a novel transcription start site-capturing massively parallel reporter assay, called TSS-MPRA, that uses a dual barcoding strategy to measure bi-directional transcription initiation of a single regulatory sequence. By taking advantage of our dual barcoding approach, we are also able to clone in paired promoter-enhancer elements and measure enhancer activity by TSS-MPRA, and thus simultaneously measure the promoter and enhancer activity of paired sequences in a single experiment (MEA-TSS-MPRA). Using MEA-TSS-MPRA we characterize the highly debated relationship between enhancer transcription and enhancer function. We find evidence of enhancer-promoter specificity and observe moderate correlation between enhancer transcription and function, though perturbation experiments of various transcription factor binding sites and core promoter elements bring into question whether they are functionally linked.