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Function-based identification of mammalian enhancers using site-specific integration

Nature Methods volume 11, pages 566–571 (2014)Cite this article

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Abstract

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro–differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

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Figure 1: Overview of SIF-seq.
Figure 2: SIF-seq identifies mouse and human ES cell enhancers.
Figure 3: Validating mouse ES cell enhancers identified by SIF-seq.
Figure 4: SIF-seq accurately identifies cardiac enhancers.

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Acknowledgements

We thank S. Bronson (Pennsylvania State University) for the pSKB1 plasmid, A. Miyawaki (RIKEN) for the Venus gene, and R. Malmstrom, K. Singh and Z. Zhao for technical help. A.V. and L.A.P. were supported by US National Institute of Health (NIH) grants U01DE020060, R01HG003988 and U54HG006997. D.E.D. was supported by NIH grant 5T32HL098057 (to Children's Hospital Oakland Research Institute). B.G.B. was supported by NIH Bench to Bassinet Program (U01HL098179). A.K. and B.G. were supported by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research, the UK Biotechnology and Biological Sciences Research Council, and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute. Research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under US Department of Energy Contract DE-AC02-05CH11231, University of California.

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Authors and Affiliations

  1. Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Diane E Dickel, Yiwen Zhu, Alex S Nord, Jennifer A Akiyama, Veena Afzal, Ingrid Plajzer-Frick, Axel Visel & Len A Pennacchio

  2. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    John N Wylie & Benoit G Bruneau

  3. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, California, USA

    John N Wylie & Benoit G Bruneau

  4. Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

    Aileen Kirkpatrick & Berthold Göttgens

  5. Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Aileen Kirkpatrick & Berthold Göttgens

  6. Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA

    Benoit G Bruneau

  7. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA

    Benoit G Bruneau

  8. US Department of Energy Joint Genome Institute, Walnut Creek, California, USA

    Axel Visel & Len A Pennacchio

  9. School of Natural Sciences, University of California, Merced, Merced, California, USA

    Axel Visel

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Contributions

D.E.D., Y.Z., J.N.W., J.A.A., V.A., I.P.-F. and A.K. carried out experimental studies. D.E.D. and A.S.N. performed data analysis. D.E.D., B.G., B.G.B., A.V. and L.A.P. planned experiments. D.E.D., A.V. and L.A.P. wrote the manuscript.

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Correspondence to Axel Visel or Len A Pennacchio.

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Dickel, D., Zhu, Y., Nord, A. et al. Function-based identification of mammalian enhancers using site-specific integration. Nat Methods 11, 566–571 (2014). https://doi.org/10.1038/nmeth.2886

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