DNase I–hypersensitive exons colocalize with promoters and distal regulatory elements

Journal name:
Nature Genetics
Year published:
Published online


The precise splicing of genes confers an enormous transcriptional complexity to the human genome. The majority of gene splicing occurs cotranscriptionally, permitting epigenetic modifications to affect splicing outcomes. Here we show that select exonic regions are demarcated within the three-dimensional structure of the human genome. We identify a subset of exons that exhibit DNase I hypersensitivity and are accompanied by 'phantom' signals in chromatin immunoprecipitation and sequencing (ChIP-seq) that result from cross-linking with proximal promoter- or enhancer-bound factors. The capture of structural features by ChIP-seq is confirmed by chromatin interaction analysis that resolves local intragenic loops that fold exons close to cognate promoters while excluding intervening intronic sequences. These interactions of exons with promoters and enhancers are enriched for alternative splicing events, an effect reflected in cell type–specific periexonic DNase I hypersensitivity patterns. Collectively, our results connect local genome topography, chromatin structure and cis-regulatory landscapes with the generation of human transcriptional complexity by cotranscriptional splicing.

At a glance


  1. A subset of exons exhibits DNase I hypersensitivity.
    Figure 1: A subset of exons exhibits DNase I hypersensitivity.

    (a) Fractional overlap of various genomic regions with DHS peaks showing significant overlap with exons (P = 8.3 × 10−14, n = 10 cell types; error bars, s.d.). (b) Top, frequency distribution of DNase I–cleaved reads at DHS exons (two replicates shown in different shades) relative to all exons. Plots were created with data from K562 cells and aligned with reference to the 3′ or 5′ end of the exon. Bottom, matched whole-genome sequencing was performed to discern any bias in sequencing and alignment due to the repetitive and informational content of exons and introns. We observe little bias at either DHS exons or total exons. Gray background shading indicates exon boundaries. (c) DHS peaks (black; auto-scaled signal) overlapping exons (gray; DHS exons indicated by blue dashed boxes) of the VWA7 gene showing the cell type specificity of DNase I sensitivity across 45 cell types. The relative mappability of loci is indicated below52.

  2. Combined ChIP-seq and ChIA-PET analysis shows that DHS exons interact with promoters and distal regulatory elements.
    Figure 2: Combined ChIP-seq and ChIA-PET analysis shows that DHS exons interact with promoters and distal regulatory elements.

    (a) Schematic showing how periexonic ChIP-seq enrichments indicate close spatial localization of exons and promoters. Initial formaldehyde treatment cross-links the preinitiation complex to occupied promoter and proximal exon sequences (purple) within the SH2D3A gene (left, dashed red circle). During ChIP-seq (top), immunoprecipitation of the initiating form of Pol II (hypophosphorylated at Ser2) yields sequenced reads that align to promoter and exonic sequences (right), resulting in periexonic ChIP-seq enrichment. ChIA-PET (bottom) employs an additional ligation step to join coprecipitating promoter and exon sequences in proximity. Alignment of reads derived from these ligated promoter-exon sequences spans interacting regions and confirms that the Pol II ChIP-seq signal observed at the SH2D3A exon results from close spatial proximity of the exon to the gene promoter upstream (right) within the genome's three-dimensional structure. (b) Heatmap indicating the relative enrichment of numerous transcription factors that distinguish DHS exons according to promoter-like (P), enhancer-like (E) and CTCF and/or cohesin (C) ChIP-seq signals. Marked boxes indicate proteins employed in downstream ChIA-PET validation. RPKM, reads per kilobase per million. (c) Histogram showing the frequency of various genomic regions undergoing ChIA-PET interactions with the promoter (top, blue), enhancer (middle, orange) or cohesin (bottom, green) sites as determined by coprecipitation with hypophosphorylated Pol II, H3K4me2 (ref. 27) and CTCF, respectively. DHS exons show enrichment of interactions with promoters (P = 0.0004, Mann-Whitney two-tailed test, n = 4; error bars, range), enhancer (n = 1) or CTCF and/or cohesin (P = 0.0005, Mann-Whitney two-tailed test, n = 3; error bars, range) sites. Similarly, DNase I peaks overlapping exons show enrichment of interactions with promoters (P = 0.0002, Mann-Whitney two-tailed test, n = 4; error bars, range), enhancers (n = 1) and CTCF and/or cohesin (P = 0.028, Mann-Whitney two-tailed test, n = 3; error bars, range) sites relative to total peaks. These findings validates the interactions between DHS exons and promoter or distal enhancer elements as anticipated by ChIP-seq enrichments. (d) Genome browser view showing selected examples of promoter (top), enhancer (middle) and CTCF and/or cohesin (bottom) interactions with exons as determined by matched ChIP-seq and ChIA-PET. DNase I sensitivity (black histogram) and additional supportive ChIP-seq enrichments (colored histograms) at genomic elements and the interacting exon are also indicated.

  3. Schematic of local genome folding of exons within the SPTBN4 gene.
    Figure 3: Schematic of local genome folding of exons within the SPTBN4 gene.

    (a) Genome browser view showing ChIA-PET interactions (red; opacity indicates interaction frequency) and ChIP-seq signal (red histogram) for initiating form of Pol II (hypophosphorylated at Ser2) that correspond with the complex exon structure of the SPTBN4 gene, with the DHS (red) and matched (blue) exons indicated. DNase I hypersensitivity of loci is shown (black histogram), along with selected ChIP-seq libraries that are enriched at DHS exons (orange histograms; auto-scaled to view). Dashed boxes indicate corresponding exons. (b) Proposed model of local structure of the SPTBN4 gene interpreted from integrated ChIA-PET, ChIP-seq and DNase I annotations. SPTBN4 exons sensitive to DNase I cleavage and proximal cross-linking (red, within dashed circle) are located close to a transcription factory containing initiating Pol II (red eclipse) in association with the STPBN4 core promoter (black arrow). Additional protein features, anticipated by ChIP-seq enrichments, are also found within the transcription factory (orange circles).

  4. 3C validates cell type-specific interactions between exons and promoters.
    Figure 4: 3C validates cell type–specific interactions between exons and promoters.

    (ac) 3C interaction profiles for promoter and downstream exon-containing HindIII fragments in the CAMK2G (a), MGRN1 (b) and HPCAL1 (c) genes (n = 3 replicate libraries per cell type; error bars, s.d.). For each gene, ChIA-PET interactions (red bars with frequency) and 3C interaction frequency for both MCF-7 (top red histogram) and K562 (bottom orange histogram) cells are indicated. All genes show significant enrichments (P > 0.05, unpaired t test) for 3C interaction between DHS exons (green dashed boxes) and upstream promoters in MCF-7 cells relative to K562 cells. RPM, reads per million.

  5. Association between DHS exons and cell type-specific alternative splicing.
    Figure 5: Association between DHS exons and cell type–specific alternative splicing.

    (a) Box-and-whisker plot (minimum-maximum range) indicating fractional overlap of DHS exons, matched exons and total exons with alternative splicing events (P = 6.52 × 10−131, two-tailed matched-pair t test, n = 86 cell types). (b) Top, definition of node and loop components for ChIA-PET interactions. Bottom, box-and-whisker plot indicating the fractional overlap of exons involved in chromatin interactions (within nodes) and exons within intervening regions (within loops) with alternative splicing events (P = 0.0015, two-tailed matched-pair t test, n = 4 library replicates). (c) Box-and-whisker plot showing fold enrichment for exon inclusion frequency for DHS exons and matched exons (P = 0.0003, Wilcoxon matched-pair signed-rank test, n = 12 cell types) relative to the background for all exons. (d) Box-and-whisker plot showing the fold enrichment of exon inclusion frequency for cell type–specific DHS exons relative to cell type–specific non-DHS exons (P = 7.81 × 10−6, Wilcoxon matched-pair signed-rank test, n = 12 cell types) relative to the background for all exons. (e) Box-and-whisker plot showing differential DHS exon usage between paired cell types (P = 1.56 × 10−6, Wilcoxon matched-pair signed-rank test, n = 5 randomly paired cell types). (f) Box-and-whisker plot showing fractional overlap of DHS exons with promoter-like, enhancer-like and insulator-like features with alternative splicing events (P = 0.0235, ANOVA, n = 8 cell types). In df, schematics (top) show the locations of DHS exons within genes.

Accession codes

Primary accessions

Gene Expression Omnibus

Sequence Read Archive


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Author information


  1. Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

    • Tim R Mercer &
    • Michael B Clark
  2. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

    • Tim R Mercer &
    • Lars K Nielsen
  3. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.

    • Stacey L Edwards
  4. SE Queensland Institute of Medical Research, Brisbane, Queensland, Australia.

    • Stacey L Edwards
  5. Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington, USA.

    • Shane J Neph,
    • Hao Wang,
    • Andrew B Stergachis,
    • Sam John,
    • Richard Sandstrom &
    • John A Stamatoyannopoulos
  6. Genome Institute of Singapore, Singapore.

    • Guoliang Li,
    • Kuljeet S Sandhu &
    • Yijun Ruan
  7. Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

    • John S Mattick
  8. Vincent's Clinical School, University of New South Wales, Kensington, New South Wales, Australia.

    • John S Mattick


T.R.M., G.L. and S.J.N. performed bioinformatic analysis. S.L.E. performed 3C analysis. G.L., K.S.S. and Y.R. performed ChIA-PET analysis. A.B.S., H.W., S.J. and R.S. performed native ChIP-seq and whole-genome sequencing. T.R.M., S.J.N., M.B.C., L.K.N., Y.R., J.S.M. and J.A.S. prepared the manuscript.

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