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Genetic dissection of the α-globin super-enhancer in vivo

Nature Genetics volume 48, pages 895–903 (2016)Cite this article

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Abstract

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

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Figure 1: The α-globin regulatory region typifies a super-enhancer in erythroid cells.
Figure 2: Erythroid super-enhancer constituents vary in transcription factor binding and chromatin signature.
Figure 3: Enhancer assays of individual elements in the α-globin super-enhancer.
Figure 4: Hematological impact of single and double enhancer knockouts.
Figure 5: α-globin gene transcription in single- and double-enhancer knockouts.
Figure 6: Analysis of chromatin structure in the α-globin super-enhancer.

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References

  1. Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Ing-Simmons, E. et al. Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin. Genome Res. 25, 504–513 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Qian, J. et al. B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell 159, 1524–1537 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Siersbæk, R. et al. Transcription factor cooperativity in early adipogenic hotspots and super-enhancers. Cell Rep. 7, 1443–1455 (2014).

    Article  PubMed  Google Scholar 

  7. Vahedi, G. et al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature 520, 558–562 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pott, S. & Lieb, J.D. What are super-enhancers? Nat. Genet. 47, 8–12 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. Kvon, E.Z. Using transgenic reporter assays to functionally characterize enhancers in animals. Genomics 106, 185–192 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Hosseini, M. et al. Causes and consequences of chromatin variation between inbred mice. PLoS Genet. 9, e1003570 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    CAS  PubMed  Google Scholar 

  14. Kowalczyk, M.S. et al. Intragenic enhancers act as alternative promoters. Mol. Cell 45, 447–458 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Dowen, J.M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hughes, J.R. et al. Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences. Proc. Natl. Acad. Sci. USA 102, 9830–9835 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Kothary, R. et al. Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 105, 707–714 (1989).

    CAS  PubMed  Google Scholar 

  19. Anguita, E. et al. Deletion of the mouse α-globin regulatory element (HS −26) has an unexpectedly mild phenotype. Blood 100, 3450–3456 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Dölken, L. et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–1972 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Geiss, G.K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Davies, J.O.J. et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat. Methods 13, 74–80 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Thurman, R.E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Grosveld, F., van Assendelft, G.B., Greaves, D.R. & Kollias, G. Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell 51, 975–985 (1987).

    Article  CAS  PubMed  Google Scholar 

  26. Parker, S.C. et al. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. Proc. Natl. Acad. Sci. USA 110, 17921–17926 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang, J. et al. Dynamic control of enhancer repertoires drives lineage and stage-specific transcription during hematopoiesis. Dev. Cell 36, 9–23 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Bender, M.A. et al. The hypersensitive sites of the murine β-globin locus control region act independently to affect nuclear localization and transcriptional elongation. Blood 119, 3820–3827 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Frankel, N. et al. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466, 490–493 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Perry, M.W., Boettiger, A.N., Bothma, J.P. & Levine, M. Shadow enhancers foster robustness of Drosophila gastrulation. Curr. Biol. 20, 1562–1567 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hong, J.W., Hendrix, D.A. & Levine, M.S. Shadow enhancers as a source of evolutionary novelty. Science 321, 1314 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Villar, D. et al. Enhancer evolution across 20 mammalian species. Cell 160, 554–566 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  35. McGowan, S.J., Hughes, J.R., Han, Z.P. & Taylor, S. MIG: Multi-Image Genome viewer. Bioinformatics 29, 2477–2478 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Sauka-Spengler, T. & Barembaum, M. Gain- and loss-of-function approaches in the chick embryo. Methods Cell Biol. 87, 237–256 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Mao, X., Fujiwara, Y. & Orkin, S.H. Improved reporter strain for monitoring Cre recombinase–mediated DNA excisions in mice. Proc. Natl. Acad. Sci. USA 96, 5037–5042 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fiering, S. et al. Targeted deletion of 5HS2 of the murine β-globin LCR reveals that it is not essential for proper regulation of the β-globin locus. Genes Dev. 9, 2203–2213 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Russell, E.S. & Berstein, S.E. in Biology of the Laboratory Mouse 2nd edn. (ed. Green, E.L.) Chapter 17 (Dover Publications, 1966).

  42. Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X.S. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 7, 1728–1740 (2012).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This manuscript is dedicated to the memory of Professor Bill Wood who initiated the project and died in 2014 during the course of this work. The work was supported by the Wellcome Trust (D.H.), the UK Medical Research Council (MRC) and the National Institute for Health Research Biomedical Research Centre, Oxford. L.A.P. was supported by NIDCR FaceBase grant U01DE020060NIH and by NHGRI grants R01HG003988 and U54HG006997, and research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under US Department of Energy contract DE-AC02-05CH11231, University of California.

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Author notes

  1. William G Wood: Deceased.

  2. Christian Babbs, James O J Davies, Bryony J Graham, Lars L P Hanssen, Mira T Kassouf, A Marieke Oudelaar, Jacqueline A Sharpe, Maria C Suciu, Jelena Telenius and Ruth Williams: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford, UK

    Deborah Hay, Jim R Hughes, Christian Babbs, James O J Davies, Bryony J Graham, Lars L P Hanssen, Mira T Kassouf, A Marieke Oudelaar, Jacqueline A Sharpe, Maria C Suciu, Jelena Telenius, Christina Rode, Pik-Shan Li, Len A Pennacchio, Jacqueline A Sloane-Stanley, Helena Ayyub, Sue Butler, Richard J Gibbons, Andrew J H Smith, William G Wood & Douglas R Higgs

  2. Weatherall Institute of Molecular Medicine, Oxford, UK

    Ruth Williams & Tatjana Sauka-Spengler

  3. Genomics Division, MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Len A Pennacchio

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Contributions

D.R.H. and W.G.W. conceived the project. D.R.H., J.R.H., D.H., J.O.J.D., L.L.P.H., M.C.S. and J.T. designed experiments. J.R.H., C.B., D.H., J.O.J.D., B.J.G., L.L.P.H., M.T.K., J.A.S., M.C.S., J.T., R.W., P.-S.L., J.A.S.-S., H.A., A.M.O., C.R. and S.B. performed experiments. D.R.H., J.R.H., C.B., D.H., J.O.J.D., B.J.G., L.L.P.H., M.T.K., M.C.S., J.T., R.W., J.A.S., A.M.O. and P.-S.L. analyzed data. D.R.H., J.R.H., C.B., D.H., J.O.J.D., B.J.G., L.L.P.H., M.T.K., M.C.S., J.T., R.W. and J.A.S. wrote the manuscript. L.A.P., A.J.H.S. and T.S.-S. provided reagents and expertise. D.R.H., J.R.H., R.J.G. and A.J.H.S. provided supervision.

Corresponding author

Correspondence to Douglas R Higgs.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–6. (PDF 47502 kb)

Circulating blood cells show R1 enhancer activity.

R1 enhancer activity in circulating blood cells, captured using a Red Epic camera attached to an Olympus MVX10 microscope, recorded at 240 frames per second at 1,280 iso, 20× magnification. (MP4 1799 kb)

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Hay, D., Hughes, J., Babbs, C. et al. Genetic dissection of the α-globin super-enhancer in vivo. Nat Genet 48, 895–903 (2016). https://doi.org/10.1038/ng.3605

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