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Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification


Sequencing of DNase I hypersensitive sites (DNase-seq) is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize performance of DNase-seq. Sequencing short fragments of 50–100 base pairs (bp) that accumulate in long internucleosome linker regions was more efficient for identifying transcription factor binding sites compared to sequencing longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy via generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNase I cutting bias, we found a strong effect that varied over more than two orders of magnitude. This indicates that the nucleotide-resolution cleavage patterns at many transcription factor binding sites are derived from intrinsic DNase I cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNase I hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations.

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Figure 1: Effect of digestion level and fragment size on recovery of known TF binding sites.
Figure 2: Nucleosome-positioning effects on DNase-seq results.
Figure 3: Pair-end sequencing of DHS sites.
Figure 4: CTCF footprint.
Figure 5: DNase I cleavage bias as revealed by AR and P53 binding.
Figure 6: Predicting TF binding from DNase-seq tag count and footprint score.

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  1. 1

    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  Article  Google Scholar 

  2. 2

    Song, L. et al. Open chromatin defined by DNase I and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res. 21, 1757–1767 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Boyle, A.P. et al. High-resolution genome-wide in vivo footprinting of diverse transcription factors in human cells. Genome Res. 21, 456–464 (2011).

    CAS  Article  Google Scholar 

  4. 4

    Degner, J.F. et al. DNase I sensitivity QTLs are a major determinant of human expression variation. Nature 482, 390–394 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Maurano, M.T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Voss, T.C. et al. Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism. Cell 146, 544–554 (2011).

    CAS  Article  Google Scholar 

  9. 9

    Ling, G., Sugathan, A., Mazor, T., Fraenkel, E. & Waxman, D.J. Unbiased, genome-wide in vivo mapping of transcriptional regulatory elements reveals sex differences in chromatin structure associated with sex-specific liver gene expression. Mol. Cell Biol. 30, 5531–5544 (2010).

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    He, H.H. et al. Differential DNase I hypersensitivity reveals factor-dependent chromatin dynamics. Genome Res. 22, 1015–1025 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Tewari, A.K. et al. Chromatin accessibility reveals insights into androgen receptor activation and transcriptional specificity. Genome Biol. 13, R88 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  14. 14

    He, H.H. et al. Nucleosome dynamics define transcriptional enhancers. Nat. Genet. 42, 343–347 (2010).

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Luger, K., Dechassa, M.L. & Tremethick, D.J. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat. Rev. Mol. Cell Biol. 13, 436–447 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Lazarovici, A. et al. Probing DNA shape and methylation state on a genomic scale with DNase I. Proc. Natl. Acad. Sci. USA 110, 6376–6381 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Campbell, V.W. & Jackson, D.A. The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA. J. Biol. Chem. 255, 3726–3735 (1980).

    CAS  PubMed  Google Scholar 

  19. 19

    Grontved, L. et al. Rapid genome-scale mapping of chromatin accessibility in tissue. Epigenetics Chromatin 5, 10 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Matys, V. et al. TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34, D108–D110 (2006).

    CAS  Article  Google Scholar 

  21. 21

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

  22. 22

    Zhang, Y., Shin, H., Song, J.S., Lei, Y. & Liu, X.S. Identifying positioned nucleosomes with epigenetic marks in human from ChIP-Seq. BMC Genomics 9, 537 (2008).

    Article  Google Scholar 

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This work was supported by grants from the US National Institutes of Health (1R01 GM099409 to X.S.L.; 1U41 HG007000 to X.S.L. and C.A.M.; 2P50 CA090381-06 to C.A.M. and M.B.; 2R01 DK074967-06 to M.B. and X.S.L., 1K99CA172948-01 to H.H.H.); the Mazzone Award (to X.S.L.), the Department of Defense (W81XWH-10-1-0557 to H.H.H.) and the Prostate Cancer Foundation (to M.B.).

Author information




H.H.H., C.A.M., H.L., X.S.L. and M.B. designed the experiments and wrote the manuscript. M.-W.C. and H.H.H. performed the experiments with the help from Y.L., P.K.R. and T.F. C.A.M., S.S.H. and H.H.H. conducted the data analysis with the help from C.Z. and H.X.

Corresponding authors

Correspondence to Henry Long or X Shirley Liu or Myles Brown.

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The authors declare no competing financial interests.

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Supplementary Figures 1–21, Supplementary Tables 1–3 and Supplementary Protocol (PDF 5336 kb)

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He, H., Meyer, C., Hu, S. et al. Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification. Nat Methods 11, 73–78 (2014).

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