Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

Abstract

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 facilitates the systematic dissection of transcriptional regulatory elements. In MPRA, microarray-synthesized DNA regulatory elements and unique sequence tags are cloned into plasmids to generate a library of reporter constructs. These constructs are transfected into cells and tag expression is assayed by high-throughput sequencing. We apply MPRA to compare >27,000 variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon-β 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 design enhancer variants that optimize potentially conflicting objectives, such as maximizing induced activity while minimizing basal activity.

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Figure 1: Overview of MPRA.
Figure 2: Single-hit scanning mutagenesis of the cAMP-responsive enhancer.
Figure 3: Single-hit scanning mutagenesis of the virus-inducible IFNB enhancer.
Figure 4: Multi-hit sampling mutagenesis of the cAMP-responsive enhancer.
Figure 5: Multi-hit sampling mutagenesis of the virus-inducible IFNB enhancer.
Figure 6: Model-based optimization.

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Acknowledgements

The authors would like to thank E.M. LeProust and S. Chen of Agilent for oligonucleotide library synthesis, R.P. Deering for assistance with Sendai virus infections and the staff of the Broad Institute and the Bauer Core facilities for assistance with data generation. This project was supported by funds from the Broad Institute, the Harvard Stem Cell Institute (T.S.M.), National Human Genome Research Institute grant R01HG004037 (M.K.), the Simons Center for Quantitative Biology at Cold Spring Harbor Laboratory (J.B.K.), National Science Foundation (NSF) grant PHY-0957573 (C.G.C., T.T.) and NSF grant PHY-1022140 (A. Mur.).

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A. Mel., X.Z., P.R., A.G. and T.S.M. developed MPRA and performed the molecular biology experiments. L.W. cultured the cells, and performed the 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.

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Correspondence to Tarjei S Mikkelsen.

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A patent application describing ideas presented in this article has been filed by the Broad Institute.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 5,6, Supplementary Notes and Supplementary Figs. 1–10 (PDF 5994 kb)

Supplementary Table 1

CRE variants (XLSX 5145 kb)

Supplementary Table 2

IFNB variants (XLSX 3389 kb)

Supplementary Table 3

CRE mutagenesis/models (XLSX 39 kb)

Supplementary Table 4

IFNB mutagenesis/models (XLSX 36 kb)

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Melnikov, A., Murugan, A., Zhang, X. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol 30, 271–277 (2012). https://doi.org/10.1038/nbt.2137

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