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High-resolution mapping of transcription factor binding sites on native chromatin

Abstract

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease–digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

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Figure 1: Identification of Reb1 binding sites on native chromatin.
Figure 2: ORGANIC TF binding sites have characteristic motifs.
Figure 3: High sensitivity and specificity of ORGANIC profiling of TFs.
Figure 4: ORGANIC sites are stably bound in vivo and are conserved throughout Saccharomyces evolution.
Figure 5: ORGANIC profiling identifies TF binding sites in inaccessible chromatin.
Figure 6: ORGANIC profiling of Drosophila GAGA factor (GAF) and Pipsqueak (Psq) DNA-binding proteins.

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Gene Expression Omnibus

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Gene Expression Omnibus

References

  1. Solomon, M.J. & Varshavsky, A. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82, 6470–6474 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ren, B. et al. Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309 (2000).

    CAS  PubMed  Google Scholar 

  3. Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).

    CAS  PubMed  Google Scholar 

  4. Zentner, G.E. & Henikoff, S. Surveying the epigenomic landscape, one base at a time. Genome Biol. 13, 250 (2012).

    PubMed  PubMed Central  Google Scholar 

  5. O'Neill, L.P. & Turner, B.M. Immunoprecipitation of native chromatin: NChIP. Methods 31, 76–82 (2003).

    CAS  PubMed  Google Scholar 

  6. Teytelman, L. et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS ONE 4, e6700 (2009).

    PubMed  PubMed Central  Google Scholar 

  7. Fan, X. & Struhl, K. Where does mediator bind in vivo? PLoS ONE 4, e5029 (2009).

    PubMed  PubMed Central  Google Scholar 

  8. Schwartz, Y.B., Kahn, T.G. & Pirrotta, V. Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes. Mol. Cell Biol. 25, 432–439 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Auerbach, R.K. et al. Mapping accessible chromatin regions using Sono-Seq. Proc. Natl. Acad. Sci. USA 106, 14926–14931 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Teytelman, L., Thurtle, D.M., Rine, J. & van Oudenaarden, A. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc. Natl. Acad. Sci. USA 110, 18602–18607 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Toth, J. & Biggin, M.D. The specificity of protein-DNA crosslinking by formaldehyde: in vitro and in Drosophila embryos. Nucleic Acids Res. 28, e4 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Jackson, V. Formaldehyde cross-linking for studying nucleosomal dynamics. Methods 17, 125–139 (1999).

    CAS  PubMed  Google Scholar 

  13. Poorey, K. et al. Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342, 369–372 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Rhee, H.S. & Pugh, B.F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147, 1408–1419 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gilfillan, G.D. et al. Limitations and possibilities of low cell number ChIP-seq. BMC Genomics 13, 645 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Roca, H. & Franceschi, R.T. Analysis of transcription factor interactions in osteoblasts using competitive chromatin immunoprecipitation. Nucleic Acids Res. 36, 1723–1730 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Teves, S.S. & Henikoff, S. Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide. Genes Dev. 25, 2387–2397 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. O'Neill, L.P. & Turner, B.M. Histone H4 acetylation distinguishes coding regions of the human genome from heterochromatin in a differentiation-dependent but transcription-independent manner. EMBO J. 14, 3946–3957 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zentner, G.E., Tsukiyama, T. & Henikoff, S. ISWI and CHD chromatin remodelers bind promoters but act in gene bodies. PLoS Genet. 9, e1003317 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Henikoff, J.G., Belsky, J.A., Krassovsky, K., MacAlpine, D.M. & Henikoff, S. Epigenome characterization at single base-pair resolution. Proc. Natl. Acad. Sci. USA 108, 18318–18323 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. MacIsaac, K.D. et al. An improved map of conserved regulatory sites for Saccharomyces cerevisiae. BMC Bioinformatics 7, 113 (2006).

    PubMed  PubMed Central  Google Scholar 

  22. Bailey, T.L. & Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36 (1994).

    CAS  PubMed  Google Scholar 

  23. Beinoraviciūte-Kellner, R., Lipps, G. & Krauss, G. In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae. FEBS Lett. 579, 4535–4540 (2005).

    PubMed  Google Scholar 

  24. Hartley, P.D. & Madhani, H.D. Mechanisms that specify promoter nucleosome location and identity. Cell 137, 445–458 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Ju, Q.D., Morrow, B.E. & Warner, J.R. REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb. Mol. Cell Biol. 10, 5226–5234 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Cho, G., Kim, J., Rho, H.M. & Jung, G. Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1. Nucleic Acids Res. 23, 2980–2987 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Hesselberth, J.R. et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat. Methods 6, 283–289 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Galas, D.J. & Schmitz, A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5, 3157–3170 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Stormo, G.D. DNA binding sites: representation and discovery. Bioinformatics 16, 16–23 (2000).

    CAS  PubMed  Google Scholar 

  31. Blanchette, M. & Tompa, M. Discovery of regulatory elements by a computational method for phylogenetic footprinting. Genome Res. 12, 739–748 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Ganapathi, M. et al. Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast. Nucleic Acids Res. 39, 2032–2044 (2011).

    CAS  PubMed  Google Scholar 

  34. Harbison, C.T. et al. Transcriptional regulatory code of a eukaryotic genome. Nature 431, 99–104 (2004).

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  35. Henikoff, S., Henikoff, J.G., Sakai, A., Loeb, G.B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460–469 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Schwendemann, A. & Lehmann, M. Pipsqueak and GAGA factor act in concert as partners at homeotic and many other loci. Proc. Natl. Acad. Sci. USA 99, 12883–12888 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. The modENCODE Consortium et al. Identification of functional elements and regulatory circuits by Drosophila modENCODE. Science 330, 1787–1797 (2010).

  38. Moorman, C. et al. Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 103, 12027–12032 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 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 10.1038/nmeth.2688 (2013).

  40. Lohman, T.M. & Mascotti, D.P. Thermodynamics of ligand-nucleic acid interactions. Methods Enzymol. 212, 400–424 (1992).

    CAS  PubMed  Google Scholar 

  41. Hager, G.L., McNally, J.G. & Misteli, T. Transcription dynamics. Mol. Cell 35, 741–753 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wilkins, R.C. & Lis, J.T. GAGA factor binding to DNA via a single trinucleotide sequence element. Nucleic Acids Res. 26, 2672–2678 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Soeller, W.C., Oh, C.E. & Kornberg, T.B. Isolation of cDNAs encoding the Drosophila GAGA transcription factor. Mol. Cell Biol. 13, 7961–7970 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhao, X., Muller, E.G. & Rothstein, R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol. Cell 2, 329–340 (1998).

    CAS  PubMed  Google Scholar 

  45. Gelbart, M.E., Rechsteiner, T., Richmond, T.J. & Tsukiyama, T. Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates. Mol. Cell Biol. 21, 2098–2106 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Furuyama, S. & Biggins, S. Centromere identity is specified by a single centromeric nucleosome in budding yeast. Proc. Natl. Acad. Sci. USA 104, 14706–14711 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Melnikova, L. et al. Interaction between the GAGA factor and Mod(mdg4) proteins promotes insulator bypass in Drosophila. Proc. Natl. Acad. Sci. USA 101, 14806–14811 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Horowitz, H. & Berg, C.A. The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis. Development 122, 1859–1871 (1996).

    CAS  PubMed  Google Scholar 

  49. Weber, C.M., Henikoff, J.G. & Henikoff, S. H2A.Z nucleosomes enriched over active genes are homotypic. Nat. Struct. Mol. Biol. 17, 1500–1507 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Stamatoyannopoulos, J.A. What does our genome encode? Genome Res. 22, 1602–1611 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhu, L.J. et al. FlyFactorSurvey: a database of Drosophila transcription factor binding specificities determined using the bacterial one-hybrid system. Nucleic Acids Res. 39, D111–D117 (2011).

    CAS  PubMed  Google Scholar 

  54. Spivak, A.T. & Stormo, G.D. ScerTF: a comprehensive database of benchmarked position weight matrices for Saccharomyces species. Nucleic Acids Res. 40, D162–D168 (2012).

    CAS  PubMed  Google Scholar 

  55. 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).

    CAS  PubMed  Google Scholar 

  56. Morrow, B.E., Ju, Q. & Warner, J.R. A bipartite DNA-binding domain in yeast Reb1p. Mol. Cell Biol. 13, 1173–1182 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Lang, W.H. & Reeder, R.H. The REB1 site is an essential component of a terminator for RNA polymerase I in Saccharomyces cerevisiae. Mol. Cell Biol. 13, 649–658 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Kharchenko, P.V. et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471, 480–485 (2011).

    CAS  PubMed  Google Scholar 

  59. Meyer, L.R. et al. The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 41, D64–D69 (2013).

    CAS  PubMed  Google Scholar 

  60. Cherry, J.M. et al. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 40, D700–D705 (2012).

    CAS  PubMed  Google Scholar 

  61. Sherman, F. Getting started with yeast. Methods Enzymol. 350, 3–41 (2002).

    CAS  PubMed  Google Scholar 

  62. Landt, S.G. et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22, 1813–1831 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J.G. Henikoff for help with data analysis, members of the S. Henikoff laboratory and L. Gabrovsek for comments on the manuscript, G. Cavalli (Institut Génétique Humaine) and C.A. Berg (University of Washington) for GAF and Psq antibodies, respectively, and the Drosophila RNAi Screening Center for cells. This work was supported by the Howard Hughes Medical Institute, grant R01 ES020116 from the US National Institutes of Health (NIH) (S.H. and K.A.), the Fred Hutchinson Cancer Research Center (FHCRC) Chromosome Metabolism and Cancer Training grant NIH 5T32 CA009657 (G.E.Z.), the European Research Council (ERC) 7th Framework Program, Marie Curie Actions IOF 300710 (G.A.O.) and the Micki & Robert Flowers ARCS Endowment from the Seattle Chapter of the ARCS Foundation (S.K.).

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Contributions

S.H. conceived of the strategy; S.K., S.H. and G.E.Z. designed the yeast experiments; K.A. and G.A.O. designed and performed the Drosophila experiments; S.K. performed the yeast experiments and yeast analysis; S.K. and G.A.O. performed the Drosophila analysis; and S.K. wrote the paper.

Corresponding author

Correspondence to Steven Henikoff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17 and Supplementary Results (PDF 7038 kb)

Supplementary Table 1

Peaks called for ORGANIC datasets reported in this study. Reb1: 2.5 min MNase/80mM NaCl/len50, 10 min MNase/80mM NaCl/len50, 10 min MNase/150mM NaCl/len50, 10 min MNase/600mM NaCl/len50; Abf1: 2.5 min MNase/80mM NaCl/len50, 10 min MNase/80mM NaCl/len50, 10 min MNase/600mM NaCl/len50; Psq len25 and GAF len25. Thresholds used for peak calling (see Online Methods) are indicated. (XLS 1017 kb)

Supplementary Table 2

Log-odds position-specific scoring matrices from S. cerevisiae ORGANIC experiments. For a given log-odds entry (row,column) in the matrix, row specifies the nucleotide and column the position in the motif. (XLS 27 kb)

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Kasinathan, S., Orsi, G., Zentner, G. et al. High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods 11, 203–209 (2014). https://doi.org/10.1038/nmeth.2766

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