Coupling transcription factor occupancy to nucleosome architecture with DNase-FLASH

Journal name:
Nature Methods
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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.

At a glance


  1. DNase-FLASH for the parallel detection of TF binding and nucleosome positioning in vivo.
    Figure 1: DNase-FLASH for the parallel detection of TF binding and nucleosome positioning in vivo.

    (a) DNase I digestion of chromatin: arrows indicate cleavage sites, nucleosomes are shown in orange, and transcription factors as red ovals. NCP, nucleosome core particle. (b) Outline of the DNase-FLASH method. (c) Lengths of DNase I fragments overlapping the top 10% most accessible DHSs or nucleosome locations. Dotted lines demarcate the size ranges used for the stratification of fragments. Arrows highlight the 10.4 bp periodicity. (df) DHSs in TSS-proximal (d) and TSS-distal (e,f) configurations. Nucleosome positioning (blue) DHS (red) and consensus motifs for 5 TFs are shown (d). Dotted lines correspond to the density of the histone modifications H3K4me3 and histone H2A.Z measured by native ChIP-seq of MNase-digested chromatin from the same cell type. (e,f) The cis-regulatory architecture surrounding distal binding sites of DLX5 (e) and CTCF (f).

  2. DNase I fragment length parallels TF occupancy.
    Figure 2: DNase I fragment length parallels TF occupancy.

    (a) Heatmap of the per-nucleotide cleavages in ±50 bp of 32,426 predicted CTCF binding sites in accessible chromatin. Each row of the heat map corresponds to one predicted CTCF binding site and columns correspond to each nucleotide ±50 bp surrounding the TF-recognition sequence. Rows are sorted by decreasing cleavage density in the ±25 bp window surrounding the motif instance. The binding consensus sequence is displayed at the top. (b) Mean fragment lengths overlapping each CTCF motif: each box corresponds to 10% of the predicted CTCF binding sites ordered as in a. Dashed red line represents the mean fragment length of all fragments detected irrespective of binding-site motif. (c,d) Heat maps of the density of TF-derived (≤125-bp) fragments (c) and nucleosome-derived (126–185-bp) fragments (d) ± 500-bp surrounding predicted CTCF binding sites ordered as in a.

  3. Transcription factors position nucleosomes and organize chromatin structure in regulatory regions.
    Figure 3: Transcription factors position nucleosomes and organize chromatin structure in regulatory regions.

    (a) Density of DNase I fragments mapped by fragment length, relative to the position of the predicted binding site (motif). Red box corresponds to the consensus CTCF motif. NCP, nucleosome core particle. (b) Density of TF binding sites predicted by the software FIMO (find individual motif occurrences) (TRANSFAC database (BioBase), FIMO motif occurrence threshold of P < 10−5) around the 10% most accessible nucleosomes. NCP, ~73 bp of DNA wrapped around the nucleosome core particle relative to the dyad axis; linker, ~35 bp of linker DNA. Dashed red line marks average motif density in DHS genome-wide. Average G+C content of the genomic sequence underlying these regions was measured in 21-bp intervals. (ce) Heatmaps showing positioning of nucleosomes ± 1 kb surrounding the predicted binding sites for CTCF (c), AP-1 (d) and USF-1 (e). Rows indicate the location of nucleosomes identified by the density of large fragments and are ordered by decreasing DNase I cleavage with ± 25 bp surround the TF recognition sequence.

  4. Boundary TFs demarcate the regulatory DNA-nucleosome interface.
    Figure 4: Boundary TFs demarcate the regulatory DNA–nucleosome interface.

    (ad) Distances between +1 nucleosomes at unidirectional promoters and the predicted motif instances for CREB-ATF family, YY1, NRF1 and NF-Y. Dashed lines denote the boundaries of +1 nucleosomes.

  5. Nucleosome organization, TSS selection and promoter activity.
    Figure 5: Nucleosome organization, TSS selection and promoter activity.

    (ac) Example promoters demonstrating the relationship of the +1 nucleosome and the annotated TSS. Gray boxes indicate nucleosomes computationally identified from the 126–185-bp fragments. (d) Aggregate DNase I–digest fragment density profiles ± 1 kb from +1 nucleosomes grouped into quartiles on the basis of signal intensity (25% most accessible in '25th percentile' to 25% least accessible in '100th percentile'). DHS density, ≤125-bp fragments; nucleosome density, 126–185-bp fragments. Colored ticks above the DNase I profiles show the density of annotated TSSs relative to the +1 nucleosome. (e) H3K4me3 enrichment around the +1 nucleosomes in each quartile from d, correlated with DNase I accessibility (colors as in d; box represents the 25th percentile and 75th percentile and the dotted lines indicate 1.5× the interquartile range).

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Author information


  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


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.

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