Massively parallel characterization of transcriptional regulatory elements

The human genome contains millions of candidate cis-regulatory elements (cCREs) with cell-type-specific activities that shape both health and myriad disease states¹. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these cCREs. Here, we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of over 680,000 sequences, representing an extensive set of annotated cCREs among three cell types (HepG2, K562, and WTC11), and found 41.7% to be active. By testing sequences in both orientations, we find promoters to have strand orientation biases and their 200 nucleotide cores to function as non-cell-type-specific ‘on switches’ providing similar expression levels to their associated gene. In contrast, enhancers have weaker orientation biases, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict cCRE function and variant effects with high accuracy, delineate regulatory motifs, and model their combinatorial effects. Testing a lentiMPRA library encompassing 60,000 cCREs in all three cell types further identified factors that determine cell-type specificity. Collectively, our work provides an extensive catalog of functional cCREs in three widely used cell lines and showcases how large-scale functional measurements can be used to dissect regulatory grammar.