Rapid dissection and model-based optimization of inducible enhancers in human cells using a massively parallel reporter assay

Learning to read and write the transcriptional regulatory code is of central importance to progress in genetic analysis and engineering. Here, we describe a massively parallel reporter assay (MPRA) that enables systematic dissection of transcriptional regulatory elements by integrating microarray-based DNA synthesis and high-throughput tag sequencing. We apply MPRA to compare more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. We first show that the resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution. We then use the data to train quantitative sequence-activity models (QSAMs) of the two enhancers. We show that QSAMs from two cellular states can be combined to identify novel enhancer variants that optimize potentially conflicting objectives, such as maximizing induced activity while minimizing basal activity.