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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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).
Gross, D.S. & Garrard, W.T. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57, 159–197 (1988).
Hesselberth, J.R. et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat. Methods 6, 283–289 (2009).
Neph, S. et al. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature 489, 83–90 (2012).
Gaffney, D.J. et al. Controls of nucleosome positioning in the human genome. PLoS Genet. 8, e1003036 (2012).
Valouev, A. et al. Determinants of nucleosome organization in primary human cells. Nature 474, 516–520 (2011).
Axel, R. Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease. Biochemistry 14, 2921–2925 (1975).
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).
Staynov, D.Z. DNase I footprinting of the nucleosome in whole nuclei. Biochem. Biophys. Res. Commun. 372, 226–229 (2008).
Noll, M. Internal structure of the chromatin subunit. Nucleic Acids Res. 1, 1573–1578 (1974).
Sabo, P. et al. Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nat. Methods 3, 511–518 (2006).
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).
Thurman, R.E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).
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).
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).
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).
Kornberg, R.D. & Stryer, L. Statistical distributions of nucleosomes: nonrandom locations by a stochastic mechanism. Nucleic Acids Res. 16, 6677–6690 (1988).
Kundaje, A. et al. Ubiquitous heterogeneity and asymmetry of the chromatin environment at regulatory elements. Genome Res. 22, 1735–1747 (2012).
Rhee, H.S. & Pugh, B.F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483, 295–301 (2012).
Yamashita, R. et al. Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res. 21, 775–789 (2011).
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).
Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887–898 (2008).
Mavrich, T.N. et al. A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. Genome Res. 18, 1073–1083 (2008).
Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
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).
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).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
John, S. et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43, 264–268 (2011).
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).
Grant, C.E., Bailey, T.L. & Noble, W.S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).
Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012).
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.
The authors declare no competing financial interests.
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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