Nucleosome dynamics define transcriptional enhancers

Journal name:
Nature Genetics
Volume:
42,
Pages:
343–347
Year published:
DOI:
doi:10.1038/ng.545
Received
Accepted
Published online

Chromatin plays a central role in eukaryotic gene regulation. We performed genome-wide mapping of epigenetically marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements, including enhancers. In prostate cancer cells, where androgen receptor binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present at androgen receptor binding sites that is flanked by a pair of marked nucleosomes. A new quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response, including androgen receptor and FOXA1. More importantly, this model also correctly predicted previously unidentified binding sites for other transcription factors present after prolonged androgen stimulation, including OCT1 and NKX3-1. Therefore, quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli.

At a glance

Figures

  1. Signal distribution and nucleosome position analysis in the androgen receptor and FOXA1 binding regions identified by ChIP-chip experiments and the TSS.
    Figure 1: Signal distribution and nucleosome position analysis in the androgen receptor and FOXA1 binding regions identified by ChIP-chip experiments and the TSS.

    (ad) H3K4me2 signal distribution relative to the center of the androgen receptor (AR) motif (a,b) and FOXA1 motif (c,d) in the binding regions. The x axis represents the distance to the center of the best AR or FOXA1 motif match in a given binding site. The y axis represents normalized ChIP-Seq tag count numbers. Veh, unstimulated condition; 4 h, stimulated conditions with treatment of DHT for 4 h. (e) Distance from the AR motif to the center of the nearest nucleosome in the AR binding sites under vehicle (red) and 4 h after DHT stimulation (blue). (f) H3K4me2 and H3K4me3 signal distribution relative to the TSS.

  2. qPCR validation of the nucleosomes stabilized-destabilized around androgen receptor (AR) binding sites.
    Figure 2: qPCR validation of the nucleosomes stabilized-destabilized around androgen receptor (AR) binding sites.

    (a) Five AR binding sites near the genes TMPRSS2, STK39, KLK3, TMC6 and TRIM35. AR ChIP-chip, AR ChIP-chip signals; H3K4me2 Veh and H3K4me2 4h, H3K4me2 ChIP-Seq signals before and after 4 h of DHT treatment. Input 4 h/Veh, qPCR assay of nucleosome fold change for DHT treatment relative to vehicle; H3K4me2 4 h/Veh, qPCR assay of fold change for H3K4me2 signal for DHT treatment relative to vehicle. Error bar, s.d. Each horizontal bar represents a NPS peak region. (b) Detailed qPCR analysis of the AR binding sites near TMPRSS2 and STK39. Each horizontal bar represents a qPCR amplification region.

  3. Motif analysis in the paired nucleosome regions.
    Figure 3: Motif analysis in the paired nucleosome regions.

    (a) Flowchart of the prediction model. The formula for the NSD score is described in the Online Methods. Treatment and control refer to treatment and vehicle control conditions. Flank refers to the 200 bp of sequence centered on each flanking nucleosome, and central refers to the sequence between these regions. (b) The fraction of androgen receptor binding sites in NSD score ranked paired nucleosome bins with decreasing score (at 4 h compared to vehicle). Paired nucleosome regions are ranked by scores representing the differences in H3K4me2 tag counts before and after DHT treatment. These ranked regions are grouped into bins of 500. Shown here is the number of regions in each bin that overlap with androgen receptor ChIP-chip–enriched regions. (c) Evolutionary conservation in the vicinity of the 5,000 highest-scoring nucleosome pairs. Mean phastCons scores representing DNA sequence conservation over 17 species is plotted as a function of the distance from the midpoint between paired nucleosomes. (d) DNA sequence content associated with nucleosome positioning. The 5,000 highest-scoring paired nucleosome regions, aligned at the midpoint, were analyzed for simple DNA sequence features: the distribution of A/T mononucleotides (black), GC dinucleotides (red) or AT dinucleotides (green). (e) Logos of androgen receptor (AR), FOXA1, NKX3-1 and OCT1 motifs from TRANSFAC library. (f) The fraction of AR binding sites in score-ranked paired nucleosome bins with decreasing score (at 16 h compared to 4 h).

  4. ChIP-qPCR and gene expression analysis of NSD scoring sites.
    Figure 4: ChIP-qPCR and gene expression analysis of NSD scoring sites.

    (ac) ChIP-qPCR validation of predicted androgen receptor (AR) (a), NKX3-1 (b) and OCT1 (c) binding sites. Box plots were generated from ChIP-qPCR data obtained from three independent experiments testing 10 sites for AR, 22 sites for NKX3-1 and 9 sites for OCT1. The individual ChIP-qPCR assays are shown in Supplementary Figure 5. (d) Correlation of paired nucleosome regions with gene expression. The fraction of differentially regulated genes with paired nucleosome regions within 20 kb is shown. The top 5,000 paired nucleosome regions were selected under the conditions of DHT treatment for 4 h versus vehicle and DHT treatment for 16 h versus DHT treatment for 4 h. Differentially regulated genes were identified as described in the Online Methods. 4 h regulated, fraction of DHT 4 h versus vehicle-treated differentially regulated genes having at least one DHT 4 h versus vehicle-paired nucleosome region within 20 kb of the transcription start site. 4 h non-regulated, fraction of non-regulated genes under the same condition. 16 h regulated and 16 h non-regulated, fractions under DHT treatment for 16 h versus 4 h. (e,f) Correlation of score and number of NSD scoring sites and upregulated gene expression, 4 h versus vehicle treatment (e) and 16 h versus 4 h treatment (f). The x axis represents the lower bound n of the number of sites within 20 kb of the TSS of a gene, and the y axis represents the odds ratio calculated by the formula (upregulated genes with at least n sites/non-regulated genes with at least n sites)/(all upregulated genes/all non-regulated genes). Red, green and blue dots represent the top 5,000, 10,000 and 20,000 NSD score sites, respectively.

Accession codes

Referenced accessions

GenBank/EMBL/DDBJ

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Author information

  1. These authors contributed equally to this work.

    • Housheng Hansen He &
    • Clifford A Meyer

Affiliations

  1. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts, USA.

    • Housheng Hansen He,
    • Clifford A Meyer,
    • Hyunjin Shin,
    • Yong Zhang &
    • X Shirley Liu
  2. Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA.

    • Housheng Hansen He,
    • Shannon T Bailey,
    • Qianben Wang,
    • Kexin Xu,
    • Min Ni,
    • Mathieu Lupien &
    • Myles Brown
  3. Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.

    • Gang Wei &
    • Keji Zhao
  4. Department of Biology, Carolina Center for the Genome Sciences and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.

    • Piotr Mieczkowski &
    • Jason D Lieb
  5. Present addresses: Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, Ohio State University College of Medicine, Columbus, Ohio, USA (Q.W.); School of Life Science and Technology, Tongji University, Shanghai, China (Y.Z.); and Department of Genetics, Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire, USA (M.L.).

    • Qianben Wang,
    • Yong Zhang &
    • Mathieu Lupien

Contributions

H.H.H., C.A.M., K.Z., J.D.L., X.S.L. and M.B. designed the experiments. H.H.H., S.T.B., G.W., Q.W., K.X., M.N., M.L. and P.M. performed the experiments. C.A.M., H.H.H., H.S. and Y.Z. performed data analysis. C.A.M., H.H.H., X.S.L., M.B., J.D.L. and M.L. wrote the manuscript.

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The authors declare no competing financial interests.

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    Supplementary Figures 1–7 and Supplementary Tables 1–3

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