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Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution

Nature Biotechnology volume 35, pages 136144 (2017) | Download Citation


Gene expression is controlled by enhancers that activate transcription from the core promoters of their target genes. Although a key function of core promoters is to convert enhancer activities into gene transcription, whether and how strongly they activate transcription in response to enhancers has not been systematically assessed on a genome-wide level. Here we describe self-transcribing active core promoter sequencing (STAP-seq), a method to determine the responsiveness of genomic sequences to enhancers, and apply it to the Drosophila melanogaster genome. We cloned candidate fragments at the position of the core promoter (also called minimal promoter) in reporter plasmids with or without a strong enhancer, transfected the resulting library into cells, and quantified the transcripts that initiated from each candidate for each setup by deep sequencing. In the presence of a single strong enhancer, the enhancer responsiveness of different sequences differs by several orders of magnitude, and different levels of responsiveness are associated with genes of different functions. We also identify sequence features that predict enhancer responsiveness and discuss how different core promoters are employed for the regulation of gene expression.

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We thank L. Cochella and members of the Stark group for comments on the manuscript and Life Science Editors ( for editorial support. We are grateful to P. Heine and E. Jans (MaxCyte) for help setting up efficient plasmid transfection. Deep sequencing was performed at the Vienna Biocenter Core Facilities GmbH (VBCF) Next-Generation Sequencing Unit ( 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

    • Cosmas D Arnold
    •  & Muhammad A Zabidi

    These authors contributed equally to this work.


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

    • Cosmas D Arnold
    • , Muhammad A Zabidi
    • , Michaela Pagani
    • , Martina Rath
    • , Katharina Schernhuber
    • , Tomáš Kazmar
    •  & Alexander Stark


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C.D.A., M.A.Z., and A.S. conceived the project. C.D.A., M.P., and M.R. performed the experiments with the help of K.S., and M.A.Z. the computational analyses. T.K. performed the k-mer based predictions. C.D.A., M.A.Z., and A.S. wrote the manuscript. 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 1–9

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  1. 1.

    Supplementary Table 1

    Enhancers used for STAP-seq screens. Genomic coordinates (dm3) and cloned sequences of S2 cell developmental (zfh1, sgl, ham), OSC developmental (tj) and housekeeping (ncm, ssp3) enhancers used for the respective STAP-seq screens.

  2. 2.

    Supplementary Table 2

    BACs that are contained in the focused libraries. Indicated are the coordinates and IDs of the BACs that were used to generate the focused STAP-seq libraries.

  3. 3.

    Supplementary Table 3

    Primers used for amplification of the D. pseudoobscura sequences (spike-in controls). Indicated are the primers used to amplify the sequences from the D. pseudoobscura genome, which were used to generate the STAP-seq spike-in control plasmids.

  4. 4.

    Supplementary Table 4

    Details on individual candidates for luciferase validations. Primer pairs used to amplify candidates for luciferase validation as well as their genomic coordinates, the luciferase fold change, standard deviations and enhancer-responsiveness are indicated.

  5. 5.

    Supplementary Table 5

    Number of mapped reads and eTSSs for STAP-seq screens. Reported are total mapped reads and collapsed fragments (see Methods) for all STAP-seq screens (dm3) and the respective D. pseudoobscura spike-in controls (dp3).

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