Massively parallel functional dissection of mammalian enhancers in vivo

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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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References

  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).

  3. 3

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

  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).

  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).

  6. 6

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

  7. 7

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

  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).

  9. 9

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

  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).

  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).

  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).

  13. 13

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

  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).

  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).

  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).

  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).

  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).

  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).

  20. 20

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

  21. 21

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

  22. 22

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

  23. 23

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

  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).

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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.

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.

Correspondence to Nadav Ahituv or 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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