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ChIP-Seq identification of weakly conserved heart enhancers

Nature Genetics volume 42, pages 806–810 (2010)Cite this article

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Abstract

Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme noncoding-sequence conservation has successfully predicted enhancers that are active in many tissues but has failed to identify substantial numbers of heart-specific enhancers. Here, we used ChIP-Seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over 3,000 candidate heart enhancers genome wide. Compared to enhancers active in othertissues we studied at this time point, most candidate heart enhancers were less deeply conserved in vertebrate evolution. Nevertheless, transgenic mouse assays of 130 candidate regions revealed that most function reproducibly as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary conservation of embryonic enhancers can vary depending on tissue type.

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Figure 1: Weak evolutionary conservation of candidate heart enhancers identified by p300 binding in embryos at E11.5.
Figure 2: In vivo testing of p300 heart enhancer predictions.
Figure 3: Examples of the diverse structural and cell type specificities of p300 ChIP-Seq identified cardiac enhancers.
Figure 4: Enrichment of heart p300 ChIP-Seq peaks near genes implicated in heart development.

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References

  1. Hoffman, J.I., Kaplan, S. & Liberthson, R.R. Prevalence of congenital heart disease. Am. Heart J. 147, 425–439 (2004).

    Article  Google Scholar 

  2. Lloyd-Jones, D. et al. Heart disease and stroke statistics–2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 119, 480–486 (2009).

    Article  Google Scholar 

  3. Pierpont, M.E. et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation 115, 3015–3038 (2007).

    Article  Google Scholar 

  4. Bruneau, B.G. The developmental genetics of congenital heart disease. Nature 451, 943–948 (2008).

    Article  CAS  Google Scholar 

  5. Bentham, J. & Bhattacharya, S. Genetic mechanisms controlling cardiovascular development. Ann. NY Acad. Sci. 1123, 10–19 (2008).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Visel, A., Rubin, E.M. & Pennacchio, L.A. Genomic views of distant-acting enhancers. Nature 461, 199–205 (2009).

    Article  CAS  Google Scholar 

  8. Aparicio, S. et al. Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes. Proc. Natl. Acad. Sci. USA 92, 1684–1688 (1995).

    Article  CAS  Google Scholar 

  9. Nobrega, M.A., Ovcharenko, I., Afzal, V. & Rubin, E.M. Scanning human gene deserts for long-range enhancers. Science 302, 413 (2003).

    Article  CAS  Google Scholar 

  10. de la Calle-Mustienes, E. et al. A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts. Genome Res. 15, 1061–1072 (2005).

    Article  CAS  Google Scholar 

  11. Woolfe, A. et al. Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol. 3, e7 (2005).

    Article  Google Scholar 

  12. Prabhakar, S. et al. Close sequence comparisons are sufficient to identify human cis-regulatory elements. Genome Res. 16, 855–863 (2006).

    Article  CAS  Google Scholar 

  13. Pennacchio, L.A. et al. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499–502 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Yao, T.P. et al. Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361–372 (1998).

    Article  CAS  Google Scholar 

  16. Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    Article  CAS  Google Scholar 

  17. Xi, H. et al. Identification and characterization of cell type-specific and ubiquitous chromatin regulatory structures in the human genome. PLoS Genet. 3, e136 (2007).

    Article  Google Scholar 

  18. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Valouev, A. et al. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat. Methods 5, 829–834 (2008).

    Article  CAS  Google Scholar 

  21. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).

    Article  CAS  Google Scholar 

  22. Kuhn, R.M. et al. The UCSC Genome Browser Database: update 2009. Nucleic Acids Res. 37, D755–D761 (2009).

    Article  CAS  Google Scholar 

  23. Kothary, R. et al. A transgene containing lacZ inserted into the dystonia locus is expressed in neural tube. Nature 335, 435–437 (1988).

    Article  CAS  Google Scholar 

  24. Srivastava, D. Making or breaking the heart: from lineage determination to morphogenesis. Cell 126, 1037–1048 (2006).

    Article  CAS  Google Scholar 

  25. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  Google Scholar 

  26. Schinke, M. Genomics of cardiovascular development, adaptation, and remodeling. (NHLBI Program for Genomic Applications, Harvard Medical School, Boston, Massachusetts, USA, 2009) 〈http://www.cardiogenomics.org〉.

  27. Olson, E.N. Gene regulatory networks in the evolution and development of the heart. Science 313, 1922–1927 (2006).

    Article  CAS  Google Scholar 

  28. Cooper, G.M. & Brown, C.D. Qualifying the relationship between sequence conservation and molecular function. Genome Res. 18, 201–205 (2008).

    Article  CAS  Google Scholar 

  29. Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. Cheng, Y. et al. Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif. Genome Res. 18, 1896–1905 (2008).

    Article  CAS  Google Scholar 

  32. Heintzman, N.D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).

    Article  CAS  Google Scholar 

  33. King, D.C. et al. Finding cis-regulatory elements using comparative genomics: some lessons from ENCODE data. Genome Res. 17, 775–786 (2007).

    Article  CAS  Google Scholar 

  34. Fisher, S., Grice, E.A., Vinton, R.M., Bessling, S.L. & McCallion, A.S. Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312, 276–279 (2006).

    Article  CAS  Google Scholar 

  35. Narlikar, L. et al. Genome-wide discovery of human heart enhancers. Genome Res 20, 381–392 (2010).

    Article  CAS  Google Scholar 

  36. Blanchette, M. et al. Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res. 14, 708–715 (2004).

    Article  CAS  Google Scholar 

  37. Murphy, W.J., Pringle, T.H., Crider, T.A., Springer, M.S. & Miller, W. Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res. 17, 413–421 (2007).

    Article  CAS  Google Scholar 

  38. Bininda-Emonds, O.R. et al. The delayed rise of present-day mammals. Nature 446, 507–512 (2007).

    Article  CAS  Google Scholar 

  39. Hedges, S.B. The origin and evolution of model organisms. Nat. Rev. Genet. 3, 838–849 (2002).

    Article  CAS  Google Scholar 

  40. Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L.A. VISTA Enhancer Browser–a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).

    Article  CAS  Google Scholar 

  41. Barrera, L.O. et al. Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs. Genome Res. 18, 46–59 (2008).

    Article  CAS  Google Scholar 

  42. Li, Z. et al. A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells. Proc. Natl. Acad. Sci. USA 100, 8164–8169 (2003).

    Article  CAS  Google Scholar 

  43. Blow, M.J. et al. Identification of ancient remains through genomic sequencing. Genome Res. 18, 1347–1353 (2008).

    Article  CAS  Google Scholar 

  44. Bryne, J.C. et al. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, D102–D106 (2008).

    Article  CAS  Google Scholar 

  45. Bailey, T.L. & Gribskov, M. Combining evidence using P values: application to sequence homology searches. Bioinformatics 14, 48–54 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors wish to thank R. Hosseini and S. Phouanenavong for technical support. L.A.P. and E.M.R. were supported by grant HL066681, Berkeley Program for Genomic Applications, which is funded by the National Heart, Lung, and Blood Institute. L.A.P. was also supported by grant HG003988, which is funded by the National Human Genome Research Institute. B.L.B was supported by grants HL64658 and HL89707 from the National Heart, Lung, and Blood Institute. B.R. is supported by funding from the National Human Genome Research Institute and the Ludwig Institute for Cancer Research. Research was conducted at the E.O. Lawrence Berkeley National Laboratory and was performed under the 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

    Matthew J Blow, Jennifer A Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Veena Afzal, Edward M Rubin, Axel Visel & Len A Pennacchio

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

    Matthew J Blow, Tao Zhang, Crystal Wright, Feng Chen, James Bristow, Edward M Rubin, Axel Visel & Len A Pennacchio

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

    David J McCulley & Brian L Black

  4. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    David J McCulley & Brian L Black

  5. Ludwig Institute for Cancer Research, University of California San Diego (UCSD) School of Medicine, La Jolla, California, USA

    Zirong Li & Bing Ren

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Contributions

M.J.B., A.V. and L.A.P. wrote the manuscript. All authors contributed to data collection and analysis and provided comments on the manuscript.

Corresponding authors

Correspondence to Axel Visel or Len A Pennacchio.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 5,7,8 and Supplementary Note. (PDF 4563 kb)

Supplementary Tables 1–4

List of p300 peaks from forebrain (XLS 2109 kb)

Supplementary Table 6

Reproducibility of LacZ staining in confirmed positive heart enhancers (XLS 45 kb)

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Blow, M., McCulley, D., Li, Z. et al. ChIP-Seq identification of weakly conserved heart enhancers. Nat Genet 42, 806–810 (2010). https://doi.org/10.1038/ng.650

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