Abstract

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear1,2. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.

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Acknowledgements

This work was supported by National Institutes of Health grants R01HG003988, U54HG006997, R24HL123879 and UM1HL098166 (to A.V. and L.A.P.) and the University of Basel and the Novartis Foundation for Biomedical Research (to J.L.-R.). M.O. was supported by a Swiss National Science Foundation (SNSF) fellowship. We thank B. Ren for providing access to the ChIP–seq and RNA-seq data from ENCODE; J. Doudna for providing a plasmid containing a human-optimized Cas9 gene; W. Ye and Y. Chen for sharing the image of a Shox2-deficient limb skeleton (Fig. 3b); and the members of the L.A.P., A.V. and D.E.D. groups for technical advice and comments on the manuscript, in particular C. Spurrell and E. Kvon. Research was conducted at the E. O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC02-05CH11231, University of California.

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Affiliations

  1. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA

    • Marco Osterwalder
    • , Iros Barozzi
    • , Yoko Fukuda-Yuzawa
    • , Brandon J. Mannion
    • , Sarah Y. Afzal
    • , Elizabeth A. Lee
    • , Yiwen Zhu
    • , Ingrid Plajzer-Frick
    • , Catherine S. Pickle
    • , Momoe Kato
    • , Tyler H. Garvin
    • , Quan T. Pham
    • , Anne N. Harrington
    • , Jennifer A. Akiyama
    • , Veena Afzal
    • , Diane E. Dickel
    • , Axel Visel
    •  & Len A. Pennacchio
  2. Department of Biomedicine, University of Basel, 4058 Basel, Switzerland

    • Virginie Tissières
    •  & Javier Lopez-Rios
  3. Centro Andaluz de Biología del Desarrollo, CSIC/JA/Universidad Pablo de Olavide, 41013 Seville, Spain

    • Virginie Tissières
    •  & Javier Lopez-Rios
  4. US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA

    • Axel Visel
    •  & Len A. Pennacchio
  5. School of Natural Sciences, University of California, Merced, California 95343, USA

    • Axel Visel
  6. Comparative Biochemistry Program, University of California, Berkeley, California 94720, USA

    • Len A. Pennacchio

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Contributions

M.O., D.E.D., A.V., and L.A.P. conceived the study. M.O., D.E.D., B.J.M., S.Y.A., E.A.L., Y.Z., I.P.-F., C.S.P., M.K., T.H.G., Q.T.P., A.N.H., J.A.A., and V.A. performed the genome editing and mouse phenotyping studies. I.B. and M.O. devised the computational framework, and I.B. performed the correlative analysis. V.T. performed in situ hybridization under the supervision of J.L.-R. Y.F.-Y. conducted the ChIP–seq and RNA-seq data analysis. M.O., D.E.D., A.V., and L.A.P. wrote the manuscript with input from the remaining authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Diane E. Dickel or Axel Visel or Len A. Pennacchio.

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https://doi.org/10.1038/nature25461

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