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Massively parallel functional dissection of mammalian enhancers in vivo

Nature Biotechnology volume 30, pages 265–270 (2012)Cite this article

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Abstract

The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian enhancers at single-nucleotide resolution through a massively parallel reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2–3% divergence from the wild-type sequence. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing the transcribed tags. Linear regression analysis yielded highly reproducible estimates of the effect of every possible single-nucleotide change on enhancer activity. The functional consequence of most mutations was modest, with ∼22% affecting activity by >1.2-fold and ∼3% by >2-fold. Several, but not all, positions with higher effects showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.

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Figure 1: Overview of MPFD.
Figure 2: Effect size on transcriptional activity of all possible substitution mutations in three mammalian enhancers.
Figure 3: Profiles of mutation effect size in TFBSs.
Figure 4: Distribution of effect sizes for all possible substitution mutations in three mammalian enhancers.

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Acknowledgements

We thank R. Qiu and J. Kitzman for advice on experimental strategies, and B. Cohen and D. Pe'er for helpful discussions. This work was supported in part by grants HG003988 from the National Human Genome Research Institute (L.A.P.), US National Institutes of Health (NIH) grant DP5OD009145 (D.M.W.), National Institute of General Medical Sciences (NIGMS) award number GM61390 (N.A.), National Institute of Child Health and Human Development (NICHD) grant number R01HD059862 (N.A.), the Pilot/Feasibility grant from the University of California, San Francisco Liver Center (P30 DK026743) (N.A.), AG039173 from the National Institute on Aging (J.B.H.) and a fellowship from the Achievement Rewards for College Scientists Foundation (J.B.H.). M.J.K. was supported in part by NIH Training grant T32 GM007175 and the Amgen Research Excellence in Bioengineering and Therapeutic Sciences Fellowship. R.P.S. is supported by a CIHR fellowship in the area of hepatology. Parts of the research were conducted at the E.O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC02-05CH11231, University of California. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH, NICHD, NHGRI or the NIGMS.

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Author notes

  1. Rupali P Patwardhan and Joseph B Hiatt: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Rupali P Patwardhan, Joseph B Hiatt, Choli Lee, Jennifer M Andrie, Su-In Lee & Jay Shendure

  2. Department of Biostatistics, University of Washington, Seattle, Washington, USA

    Daniela M Witten

  3. Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California San Francisco, San Francisco, California, USA

    Mee J Kim, Robin P Smith & Nadav Ahituv

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

    Dalit May & Len A Pennacchio

  5. Department of Computer Science, University of Washington, Seattle, Washington, USA

    Su-In Lee

  6. HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA

    Gregory M Cooper

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

    Len A Pennacchio

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  1. Rupali P Patwardhan
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Contributions

R.P.P., J.B.H., N.A., L.A.P. and J.S. conceived of key aspects of the project and planned experiments. R.P.P., M.J.K., R.P.S., D.M., C.L. and J.M.A. performed experiments. R.P.P., J.B.H., D.M.W. and G.M.C. analyzed the data. D.M.W. and S.-I.L. contributed guidance with statistical analyses. R.P.P., J.B.H. and J.S. wrote the manuscript. All authors commented on and revised the manuscript.

Corresponding authors

Correspondence to Nadav Ahituv, Len A Pennacchio or Jay Shendure.

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The authors declare no competing financial interests.

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Supplementary Tables 1–4, Supplementary Note, Supplementary Methods and Supplementary Figs. 1–10 (PDF 8862 kb)

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Patwardhan, R., Hiatt, J., Witten, D. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30, 265–270 (2012). https://doi.org/10.1038/nbt.2136

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