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FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells

Nature Methods volume 11, pages 559–565 (2014)Cite this article

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Abstract

Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a high-throughput functional assay for directly identifying active promoter and enhancer elements called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), which we used to simultaneously assess over 80,000 DNA fragments derived from nucleosome-free regions within the chromatin of embryonic stem cells (ESCs) and identify 6,364 active regulatory elements. Many of these represent newly discovered ESC-specific enhancers, showing enriched binding-site motifs for ESC-specific transcription factors including SOX2, POU5F1 (OCT4) and KLF4. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.

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Figure 1: Overview of FIREWACh.
Figure 2: NFR-derived DNAs correspond to accessible chromatin regions located throughout the genome.
Figure 3: NFR-GFP-LVs detect active CRMs.
Figure 4: FIREWACh elements are associated with expressed genes and correlate with chromatin marks of active promoters and enhancers.
Figure 5: Distal FIREWACh elements correlate with previously predicted ESC enhancers and act as cell-specific enhancers that can be used for TF prediction.

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Acknowledgements

We thank A. Heguy, E. Mazzoni, M. Stadtfeld, D. Smith and U. Basu-Roy for their critical reading of this manuscript. We also thank A. Mansukhani and members of the Skok laboratory for helpful discussions, and the Genome Technology Center and Flow Cytometry and Cell Sorting Center at New York University Langone Medical Center for technical assistance. This work was supported by an Empire State Stem Cell Board grant through the New York State Department of Health (NYSTEM Contract #CO24322) to L.D. and a postdoctoral fellowship from US National Institutes of Health NCI institutional training grant 1T32CA160002 awarded to M.M.

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Authors and Affiliations

  1. Department of Microbiology, New York University School of Medicine, New York, New York, USA

    Matthew Murtha, Zeynep Tokcaer-Keskin, Yatong Wang, Xiangmei Xi, Claudio Basilico & Lisa Dailey

  2. Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA

    Zuojian Tang & Stuart Brown

  3. Department of Biology and Biological Sciences, Yale University, New Haven, Connecticut, USA

    Francesco Strino & Yuval Kluger

  4. Department of Biology, New York University, New York, New York, USA

    Xi Chen & Richard Bonneau

  5. Department of Computer Science, Courant Institute of Mathematical Sciences, New York, New York, USA

    Richard Bonneau

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  1. Matthew Murtha
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Contributions

L.D. conceived of and designed the study; Y.W. constructed the NFR-GFP-LV reporter libraries and viral stocks; M.M. developed the FIREWACh technology and generated all cell lines, FACS sorting and PCR rescue and analysis of FIREWACh elements; Z.T. performed all processing and genomic alignment of Illumina data; S.B. generated the in silico DNA data sets; Z.T.-K. and X.X. performed luciferase assays; C.B. contributed to critical discussions; X.C., F.S., Y.K. and R.B. provided bioinformatic support and analyses; and M.M. and L.D. wrote the manuscript with additional contributions from X.C. and R.B.

Corresponding author

Correspondence to Lisa Dailey.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Note (PDF 1871 kb)

Supplementary Table 1

Genomic Coordinates for in silico DNAs, input NFR Library DNAs, FIREWACh Elements (XLSX 10 kb)

Supplementary Table 2

Sequencing Statistics (XLSX 4111 kb)

Supplementary Table 3

Luciferase Validated Elements (XLSX 32 kb)

Supplementary Table 4

Primer Sequences (XLSX 12 kb)

Supplementary Table 5

Datasets Used (XLSX 48 kb)

Supplementary Table 6

GREAT Analysis (XLSX 10 kb)

Supplementary Table 7

Full Motif Analysis (XLSX 2730 kb)

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Murtha, M., Tokcaer-Keskin, Z., Tang, Z. et al. FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells. Nat Methods 11, 559–565 (2014). https://doi.org/10.1038/nmeth.2885

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