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 microarraybased 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 cAMPregulated 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 sequenceactivity 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. Correspondence to: Tarjei S. Mikkelsen (tarjei@broadinstitute.org). *These authors contributed equally to this work. AUTHOR CONTRIBUTIONS A.Mel., X.Z., P.R., A.G. and T.S.M. developed MPRA and performed the molecular biology experiments. L.W. performed the cell culture, plasmid transfections and luciferase assays. A.Mur., T.T., S.F., C.G.C., J.B.K., M.K., E.S.L. and T.S.M. analyzed the data. T.S.M. wrote the main text with substantial input from all authors. C.G.C. and J.B.K. wrote the Supplementary Notes with substantial input from A.Mur and T.S.M. COMPETING FINANCIAL INTERESTS A patent application describing ideas presented in this article has been filed by the Broad Institute. ACCESSION NUMBERS All analyzed sequence data has been deposited in NCBI GEO under accession GSE31982. NIH Public Access Author Manuscript Nat Biotechnol. Author manuscript; available in PMC 2012 September 01. Published in final edited form as: Nat Biotechnol. ; 30(3): 271–277. doi:10.1038/nbt.2137. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript