Massively parallel functional dissection of mammalian enhancers in vivo


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes


Sequence Read Archive


  1. 1

    The ENCODE Project Consortium. A user's guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 9, e1001046 (2011).

  2. 2

    Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Cooper, G.M. & Shendure, J. Needles in stacks of needles: finding disease-causal variants in a wealth of genomic data. Nat. Rev. Genet. 12, 628–640 (2011).

    CAS  Article  Google Scholar 

  5. 5

    Kleinjan, D.A. & van Heyningen, V. Long-range control of gene expression: emerging mechanisms and disruption in disease. Am. J. Hum. Genet. 76, 8–32 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Noonan, J.P. & McCallion, A.S. Genomics of long-range regulatory elements. Annu. Rev. Genomics Hum. Genet. 11, 1–23 (2010).

    CAS  Article  Google Scholar 

  7. 7

    VanderMeer, J.E. & Ahituv, N. cis-regulatory mutations are a genetic cause of human limb malformations. Dev. Dyn. 240, 920–930 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Hindorff, L.A. et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc. Natl. Acad. Sci. USA 106, 9362–9367 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Patwardhan, R.P. et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat. Biotechnol. 27, 1173–1175 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Sabourin, J.C. et al. An intronic enhancer essential for tissue-specific expression of the aldolase B transgenes. J. Biol. Chem. 271, 3469–3473 (1996).

    CAS  Article  Google Scholar 

  11. 11

    Gregori, C. et al. Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator. Biochem. Biophys. Res. Commun. 176, 722–729 (1991).

    CAS  Article  Google Scholar 

  12. 12

    Gregori, C., Porteu, A., Mitchell, C., Kahn, A. & Pichard, A.L. In vivo functional characterization of the aldolase B gene enhancer. J. Biol. Chem. 277, 28618–28623 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Kim, M.J. et al. Functional characterization of liver enhancers that regulate drug-associated transporters. Clin. Pharmacol. Ther. 89, 571–578 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Dang, Q. et al. Structure of the hepatic control region of the human apolipoprotein E/C-I gene locus. J. Biol. Chem. 270, 22577–22585 (1995).

    CAS  Article  Google Scholar 

  15. 15

    Hiatt, J.B., Patwardhan, R.P., Turner, E.H., Lee, C. & Shendure, J. Parallel, tag-directed assembly of locally derived short sequence reads. Nat. Methods 7, 119–122 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Zhang, G., Budker, V. & Wolff, J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10, 1735–1737 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Kel, A.E. et al. MATCH: A tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Res. 31, 3576–3579 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Loots, G.G. et al. Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. Science 288, 136–140 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Margulies, E.H. et al. Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome. Genome Res. 17, 760–774 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Visel, A. et al. Ultraconservation identifies a small subset of extremely constrained developmental enhancers. Nat. Genet. 40, 158–160 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Blow, M.J. et al. ChIP-Seq identification of weakly conserved heart enhancers. Nat. Genet. 42, 806–810 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Schmidt, D. et al. Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding. Science 328, 1036–1040 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Cooper, G.M. et al. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 15, 901–913 (2005).

    CAS  Article  Google Scholar 

  24. 24

    The 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  25. 25

    Botstein, D. & Shortle, D. Strategies and applications of in vitro mutagenesis. Science 229, 1193–1201 (1985).

    CAS  Article  Google Scholar 

Download references


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.

Author information




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 or Len A Pennacchio or Jay Shendure.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Supplementary Data (ZIP 99 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Patwardhan, R., Hiatt, J., Witten, D. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30, 265–270 (2012).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing