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Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment

Abstract

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.

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Figure 1: Interaction profile viewed fromthe α-globin gene promoters.
Figure 2: Validation of the Capture-C technique.
Figure 3: Characterization of the Tal1 and Slc25a37 loci.
Figure 4: Using Capture-C to determine the effect of distal SNPs on gene regulation.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  5. de Laat, W. & Dekker, J. 3C-based technologies to study the shape of the genome. Methods 58, 189–191 (2012).

    Article  CAS  Google Scholar 

  6. Sajan, S.A. & Hawkins, R.D. Methods for identifying higher-order chromatin structure. Annu. Rev. Genomics Hum. Genet. 13, 59–82 (2012).

    Article  CAS  Google Scholar 

  7. Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

    Article  CAS  Google Scholar 

  8. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).

    Article  CAS  Google Scholar 

  9. Sexton, T. et al. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148, 458–472 (2012).

    Article  CAS  Google Scholar 

  10. van de Werken, H.J. et al. Robust 4C-seq data analysis to screen for regulatory DNA interactions. Nat. Methods 9, 969–972 (2012).

    Article  CAS  Google Scholar 

  11. Schaub, M.A., Boyle, A.P., Kundaje, A., Batzoglou, S. & Snyder, M. Linking disease associations with regulatory information in the human genome. Genome Res. 22, 1748–1759 (2012).

    Article  CAS  Google Scholar 

  12. Bainbridge, M.N. et al. Whole exome capture in solution with 3 Gbp of data. Genome Biol. 11, R62 (2010).

    Article  Google Scholar 

  13. Göndör, A., Rougier, C. & Ohlsson, R. High-resolution circular chromosome conformation capture assay. Nat. Protoc. 3, 303–313 (2008).

    Article  Google Scholar 

  14. Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. De Gobbi, M. et al. Tissue-specific histone modification and transcription factor binding in α globin gene expression. Blood 110, 4503–4510 (2007).

    Article  CAS  Google Scholar 

  17. Vavouri, T., McEwen, G.K., Woolfe, A., Gilks, W.R. & Elgar, G. Defining a genomic radius for long-range enhancer action: duplicated conserved non-coding elements hold the key. Trends Genet. 22, 5–10 (2006).

    Article  CAS  Google Scholar 

  18. Wallace, H.A. et al. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell 128, 197–209 (2007).

    Article  CAS  Google Scholar 

  19. Lower, K.M. et al. Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition. Proc. Natl. Acad. Sci. USA 106, 21771–21776 (2009).

    Article  CAS  Google Scholar 

  20. Vernimmen, D. et al. Chromosome looping at the human α-globin locus is mediated via the major upstream regulatory element (HS-40). Blood 114, 4253–4260 (2009).

    Article  CAS  Google Scholar 

  21. Baù, D. et al. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat. Struct. Mol. Biol. 18, 107–114 (2011).

    Article  Google Scholar 

  22. Hughes, J.R. et al. High-resolution analysis of cis-acting regulatory networks at the α-globin locus. Phil. Trans. R. Soc. Lond. B 368, 20120361 (2013).

    Article  Google Scholar 

  23. Vernimmen, D., De Gobbi, M., Sloane-Stanley, J.A., Wood, W.G. & Higgs, D.R. Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression. EMBO J. 26, 2041–2051 (2007).

    Article  CAS  Google Scholar 

  24. Ferreira, R. et al. Impaired in vitro erythropoiesis following deletion of the Scl (Tal1) +40 enhancer is largely compensated for in vivo despite a significant reduction in expression. Mol. Cell. Biol. 33, 1254–1266 (2013).

    Article  CAS  Google Scholar 

  25. Ogilvy, S. et al. The SCL +40 enhancer targets the midbrain together with primitive and definitive hematopoiesis and is regulated by SCL and GATA proteins. Mol. Cell. Biol. 27, 7206–7219 (2007).

    Article  CAS  Google Scholar 

  26. Göttgens, B. et al. The scl +18/19 stem cell enhancer is not required for hematopoiesis: identification of a 5′ bifunctional hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1. Mol. Cell. Biol. 24, 1870–1883 (2004).

    Article  Google Scholar 

  27. Flygare, J., Rayon Estrada, V., Shin, C., Gupta, S. & Lodish, H.F. HIF1α synergizes with glucocorticoids to promote BFU-E progenitor self-renewal. Blood 117, 3435–3444 (2011).

    Article  CAS  Google Scholar 

  28. Shaw, G.C. et al. Mitoferrin is essential for erythroid iron assimilation. Nature 440, 96–100 (2006).

    Article  CAS  Google Scholar 

  29. Amigo, J.D. et al. Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis. Mol. Cell. Biol. 31, 1344–1356 (2011).

    Article  CAS  Google Scholar 

  30. Sanyal, A., Lajoie, B.R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Kang, J.H. et al. Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5′-phosphate oxidase. Eur. J. Biochem. 271, 2452–2461 (2004).

    Article  CAS  Google Scholar 

  33. Neph, S. et al. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature 489, 83–90 (2012).

    Article  CAS  Google Scholar 

  34. Donnelly, P. Progress and challenges in genome-wide association studies in humans. Nature 456, 728–731 (2008).

    Article  CAS  Google Scholar 

  35. Bush, W.S. & Moore, J.H. Chapter 11: genome-wide association studies. PLoS Comput. Biol. 8, e1002822 (2012).

    Article  CAS  Google Scholar 

  36. Pennacchio, L.A., Bickmore, W., Dean, A., Nobrega, M.A. & Bejerano, G. Enhancers: five essential questions. Nat. Rev. Genet. 14, 288–295 (2013).

    Article  CAS  Google Scholar 

  37. Kolovos, P., Knoch, T.A., Grosveld, F.G., Cook, P.R. & Papantonis, A. Enhancers and silencers: an integrated and simple model for their function. Epigenetics Chromatin 5, 1 (2012).

    Article  CAS  Google Scholar 

  38. Spivak, J.L., Toretti, D. & Dickerman, H.W. Effect of phenylhydrazine-induced hemolytic anemia on nuclear RNA polymerase activity of the mouse spleen. Blood 42, 257–266 (1973).

    CAS  Google Scholar 

  39. Nichols, J., Evans, E.P. & Smith, A.G. Establishment of germ-line–competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110, 1341–1348 (1990).

    CAS  Google Scholar 

  40. Hagège, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat. Protoc. 2, 1722–1733 (2007).

    Article  Google Scholar 

  41. Stadhouders, R. et al. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions. Nat. Protoc. 8, 509–524 (2013).

    Article  CAS  Google Scholar 

  42. Kassouf, M.T. et al. Genome-wide identification of TAL1's functional targets: insights into its mechanisms of action in primary erythroid cells. Genome Res. 20, 1064–1083 (2010).

    Article  CAS  Google Scholar 

  43. Tallack, M.R. et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 20, 1052–1063 (2010).

    Article  CAS  Google Scholar 

  44. Papadopoulos, G.L. et al. GATA-1 genome-wide occupancy associates with distinct epigenetic profiles in mouse fetal liver erythropoiesis. Nucleic Acids Res. 41, 4938–4948 (2013).

    Article  CAS  Google Scholar 

  45. Zhou, Z. et al. USF and NF-E2 cooperate to regulate the recruitment and activity of RNA polymerase II in the β-globin gene locus. J. Biol. Chem. 285, 15894–15905 (2010).

    Article  CAS  Google Scholar 

  46. Horakova, A.H., Moseley, S.C., McLaughlin, C.R., Tremblay, D.C. & Chadwick, B.P. The macrosatellite DXZ4 mediates CTCF-dependent long-range intrachromosomal interactions on the human inactive X chromosome. Hum. Mol. Genet. 21, 4367–4377 (2012).

    Article  CAS  Google Scholar 

  47. 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  Google Scholar 

  48. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  Google Scholar 

  49. Kent, W.J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).

    Article  CAS  Google Scholar 

  50. Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinformatics Chapter 4, Unit 4.10 (2009).

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

    Article  CAS  Google Scholar 

  52. Stein, L.D. Using GBrowse 2.0 to visualize and share next-generation sequence data. Brief. Bioinform. 14, 162–171 (2013).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Davies and P. Piazza for technical suggestions. We thank M. Suciu, B. Graham and T. Milne for suggestions and critically reading the manuscript. We thank Z.-P. Han and J. Telenius for computational support. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070) for the generation of the sequencing data. This work was supported by the MRC (UK) and by the Blood theme within Oxford Biomedical Research Centre (which is part of the National Institute for Health Research Biomedical Research Centres scheme). We also thank EpiGeneSys for support.

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J.R.H., D.R.H. and R.G. designed experiments. J.R.H., N.R., D.H., M.L. and M.D.G. performed experiments. J.R.H. performed bioinformatic analysis. E.G. performed statistical analysis. S.M. and S.T. provided bioinformatic support. J.R.H. and D.R.H. wrote the manuscript.

Corresponding authors

Correspondence to Jim R Hughes or Douglas R Higgs.

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The authors declare no competing financial interests.

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Supplementary Figures 1–15, Supplementary Note and Supplementary Tables 1 and 2 (PDF 11216 kb)

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Hughes, J., Roberts, N., McGowan, S. et al. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat Genet 46, 205–212 (2014). https://doi.org/10.1038/ng.2871

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