Abstract

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.

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Acknowledgements

We thank T. Decker and G. Versteeg (Max F. Perutz Laboratories & University of Vienna), S. Aerts (VIB-KU Leuven), P. Svoboda (Institute of Molecular Genetics of the ASCR), P. Andersen (IMBA), P. Heine and E. Jans (MaxCyte Inc.) and J. Zuber (IMP) for helpful discussions and reagents. Deep sequencing was performed at the VBCF Next-Generation Sequencing Unit (http://vbcf.ac.at). F.M. was supported by an EMBO long-term fellowship (EMBO ALTF 491–2014). Research in the Stark group is supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 647320) and by the Austrian Science Fund (FWF, F4303-B09). Basic research at the IMP is supported by Boehringer Ingelheim GmbH and the Austrian Research Promotion Agency (FFG).

Author information

Author notes

    • Felix Muerdter
    •  & Łukasz M Boryń

    These authors contributed equally to this work.

Affiliations

  1. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, Vienna, Austria.

    • Felix Muerdter
    • , Łukasz M Boryń
    • , Ashley R Woodfin
    • , Christoph Neumayr
    • , Martina Rath
    • , Muhammad A Zabidi
    • , Michaela Pagani
    • , Vanja Haberle
    • , Tomáš Kazmar
    • , Rui R Catarino
    • , Katharina Schernhuber
    • , Cosmas D Arnold
    •  & Alexander Stark
  2. Medical University of Vienna, Vienna Biocenter (VBC), Vienna, Austria.

    • Alexander Stark

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Contributions

F.M. and L.M.B. are shared first authors; A.R.W. and C.N. are shared second authors. F.M., L.M.B., C.N., M.R., M.P., R.R.C., K.S. and C.D.A. performed experiments. L.M.B. designed and cloned all STARR-seq and luciferase vectors. A.R.W. and F.M. performed the computational analysis with the help of M.A.Z.; V.H. and T.K. analyzed the ORI sequence. F.M. and A.S. wrote the manuscript with help from all authors. A.S. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alexander Stark.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables

    Supplementary Tables 1, 3, and 5.

  4. 4.

    Supplementary Protocol 1

    STARR-seq Library preparation protocol

  5. 5.

    Supplementary Protocol 2

    STARR-seq Screening protocol

  6. 6.

    Supplementary Protocol 3

    qPCR assay to measure ISG expression in human cells

  7. 7.

    Supplementary Protocol 4

    qPCR based reporter assay on luciferase transcripts

Excel files

  1. 1.

    Supplementary Table 2

    STARR-seq peaks and shortlisted regions

  2. 2.

    Supplementary Table 4

    STARR-seq cloning sequences and oligos

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DOI

https://doi.org/10.1038/nmeth.4534

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