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Coupling transcription factor occupancy to nucleosome architecture with DNase-FLASH

Nature Methods volume 11, pages 66–72 (2014)Cite this article

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Abstract

It is currently not possible to resolve the genome-wide relationship of transcription factors (TFs) and nucleosomes at the level of individual chromatin templates despite rapidly increasing data on TF and nucleosome occupancy in the human genome. Here we describe DNase I–released fragment-length analysis of hypersensitivity (DNase-FLASH), an approach that directly couples mapping of TF occupancy, via quantification of DNA microfragments released from individual TF recognition sites in regulatory DNA, to the surrounding nucleosome architecture, via analysis of larger DNA fragments, in a single assay. DNase-FLASH enables coupling of individual TF footprints to nucleosome occupancy, identifying TFs that precisely demarcate the regulatory DNA–nucleosome interface.

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Figure 1: DNase-FLASH for the parallel detection of TF binding and nucleosome positioning in vivo.
Figure 2: DNase I fragment length parallels TF occupancy.
Figure 3: Transcription factors position nucleosomes and organize chromatin structure in regulatory regions.
Figure 4: Boundary TFs demarcate the regulatory DNA–nucleosome interface.
Figure 5: Nucleosome organization, TSS selection and promoter activity.

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References

  1. Workman, J.L., Abmayr, S.M., Cromlish, W.A. & Roeder, R.G. Transcriptional regulation by the immediate early protein of pseudorabies virus during in vitro nucleosome assembly. Cell 55, 211–219 (1988).

    Article  CAS  Google Scholar 

  2. Gross, D.S. & Garrard, W.T. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57, 159–197 (1988).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Gaffney, D.J. et al. Controls of nucleosome positioning in the human genome. PLoS Genet. 8, e1003036 (2012).

    Article  CAS  Google Scholar 

  6. Valouev, A. et al. Determinants of nucleosome organization in primary human cells. Nature 474, 516–520 (2011).

    Article  CAS  Google Scholar 

  7. Axel, R. Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease. Biochemistry 14, 2921–2925 (1975).

    Article  CAS  Google Scholar 

  8. Lutter, L.C. Kinetic analysis of deoxyribonuclease I cleavages in the nucleosome core: evidence for a DNA superhelix. J. Mol. Biol. 124, 391–420 (1978).

    Article  CAS  Google Scholar 

  9. Staynov, D.Z. DNase I footprinting of the nucleosome in whole nuclei. Biochem. Biophys. Res. Commun. 372, 226–229 (2008).

    Article  CAS  Google Scholar 

  10. Noll, M. Internal structure of the chromatin subunit. Nucleic Acids Res. 1, 1573–1578 (1974).

    Article  CAS  Google Scholar 

  11. Sabo, P. et al. Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nat. Methods 3, 511–518 (2006).

    Article  CAS  Google Scholar 

  12. Jin, C. et al. H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions. Nat. Genet. 41, 941–945 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Stamatoyannopoulos, J.A., Goodwin, A., Joyce, T. & Lowrey, C.H. NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region. EMBO J. 14, 106–116 (1995).

    Article  CAS  Google Scholar 

  15. Fu, Y., Sinha, M., Peterson, C.L. & Weng, Z. The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome. PLoS Genet. 4, e1000138 (2008).

    Article  Google Scholar 

  16. Cuddapah, S. et al. Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains. Genome Res. 19, 24–32 (2009).

    Article  CAS  Google Scholar 

  17. Kornberg, R.D. & Stryer, L. Statistical distributions of nucleosomes: nonrandom locations by a stochastic mechanism. Nucleic Acids Res. 16, 6677–6690 (1988).

    Article  CAS  Google Scholar 

  18. Kundaje, A. et al. Ubiquitous heterogeneity and asymmetry of the chromatin environment at regulatory elements. Genome Res. 22, 1735–1747 (2012).

    Article  CAS  Google Scholar 

  19. Rhee, H.S. & Pugh, B.F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483, 295–301 (2012).

    Article  CAS  Google Scholar 

  20. Yamashita, R. et al. Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res. 21, 775–789 (2011).

    Article  CAS  Google Scholar 

  21. Zhang, Y. et al. Intrinsic histone-DNA interactions are not the major determinant of nucleosome positions in vivo. Nat. Struct. Mol. Biol. 16, 847–852 (2009).

    Article  CAS  Google Scholar 

  22. Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887–898 (2008).

    Article  CAS  Google Scholar 

  23. Mavrich, T.N. et al. A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. Genome Res. 18, 1073–1083 (2008).

    Article  CAS  Google Scholar 

  24. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  Google Scholar 

  25. Bernstein, B.E. et al. Methylation of histone H3 Lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. USA 99, 8695–8700 (2002).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  28. John, S. et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43, 264–268 (2011).

    Article  CAS  Google Scholar 

  29. Wingender, E., Dietze, P., Karas, H. & Knüppel, R. TRANSFAC: a database on transcription factors and their DNA binding sites. Nucleic Acids Res. 24, 238–241 (1996).

    Article  CAS  Google Scholar 

  30. Grant, C.E., Bailey, T.L. & Noble, W.S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).

    Article  CAS  Google Scholar 

  31. Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

J.V. is supported by a US National Science Foundation Graduate Research Fellowship under grant DGE-071824. This work was supported by US National Institutes of Health NHGRI grants U54HG004592 and U54HG007010 to J.A.S.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Jeff Vierstra, Hao Wang, Sam John, Richard Sandstrom & John A Stamatoyannopoulos

  2. Department of Medicine, Division of Oncology, University of Washington, Seattle, Washington, USA

    John A Stamatoyannopoulos

Authors
  1. Jeff Vierstra
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  2. Hao Wang
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  3. Sam John
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  4. Richard Sandstrom
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  5. John A Stamatoyannopoulos
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Contributions

J.V. and J.A.S. designed the experiments. J.V. performed the DNase I experiments and analyzed the data. H.W. produced the MNase data. R.S. and S.J. assisted in data analysis. J.V., S.J. and J.A.S. wrote the paper.

Corresponding author

Correspondence to John A Stamatoyannopoulos.

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The authors declare no competing financial interests.

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Supplementary Figures 1–11 and Supplementary Table 1 (PDF 8647 kb)

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Vierstra, J., Wang, H., John, S. et al. Coupling transcription factor occupancy to nucleosome architecture with DNase-FLASH. Nat Methods 11, 66–72 (2014). https://doi.org/10.1038/nmeth.2713

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