Abstract

Enhancers act to regulate cell-type-specific gene expression by facilitating the transcription of target genes. In mammalian cells, active or primed enhancers are commonly marked by monomethylation of histone H3 at lysine 4 (H3K4me1) in a cell-type-specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC mass spectrometry experiments with mononucleosomes and identified multiple H3K4me1-associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments association of the chromatin-remodeling complex BAF to enhancers in vivo and that, in vitro, H3K4me1-marked nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of the BAF component BAF45C indicate that monomethylation, but not trimethylation, is accommodated by BAF45C’s H3K4-binding site. Our results suggest that H3K4me1 has an active role at enhancers by facilitating binding of the BAF complex and possibly other chromatin regulators.

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Acknowledgements

The authors thank M. Carey (UCLA) for WT and mutant histone constructs, S. Kuan and B. Li for processing of ChIP–seq samples, J. Liang and G. Hon for help and advice in SILAC mass spectrometry analysis, J. Wysocka and K. Dorighi (Stanford School of Medicine) for sharing the KMT2C/D dCD mESC line, and I. Jung for advice on ChIP–seq data analysis. We also thank T. Gahman (Ludwig Institute for Cancer Research, LICR) for arranging for peptide synthesis and A. Bobkov for assistance with isothermal titration calorimetry. The research was supported in part by 5R01GM115961. C.P.A. and H.Z. were supported by funding from LICR and NIH GM116897. A.K.S., K.D.C., H.Z. and B.R. received funding and salary support from LICR. W.W. and D.W. were supported by funding from NIH GM102362. A.L. was supported by NIH Training Grant 5T32CA009523.

Author information

Author notes

  1. Andrea Local and Hui Huang contributed equally to this work.

Affiliations

  1. Ludwig Institute for Cancer Research, La Jolla, CA, USA

    • Andrea Local
    • , Hui Huang
    • , Claudio P. Albuquerque
    • , Namit Singh
    • , Ah Young Lee
    • , Judy E. Hsia
    • , Andrew K. Shiau
    • , Kevin D. Corbett
    • , Huilin Zhou
    •  & Bing Ren
  2. Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA

    • Hui Huang
  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA

    • Wei Wang
    • , Chaochen Wang
    •  & Dong Wang
  4. National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD, USA

    • Kai Ge
  5. Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine, La Jolla, CA, USA

    • Kevin D. Corbett
    • , Huilin Zhou
    •  & Bing Ren
  6. Center for Epigenomics, Institute of Genomic Medicine, La Jolla, CA, USA

    • Bing Ren
  7. Aptose Biosciences, Inc., San Diego, CA, USA

    • Andrea Local

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Contributions

A.L. and B.R. conceived the study and prepared the manuscript. A.L. designed and carried out the SILAC experiments, nucleosome pulldown experiments, and ChIP–seq experiments and prepared the manuscript. H.H. performed H3K4me2 ChIP–seq analysis and all experiments with the dCD cell lines. A.Y.L. prepared sequencing libraries. C.P.A. ran the mass spectrometry samples in the laboratory of H.Z. and provided expertise in mass spectrometry analysis. H.H. performed ChIP–seq data analysis. C.W. and K.G. provided KMT2C/D DKO mESCs and shared expertise and data. W.W. and D.W. designed and executed the remodeling assays. A.K.S. designed and supplied H3 tail peptides and, along with J.E.H., provided advice on their use in biochemical studies. N.S. purified BAF45C, performed H3 tail peptide binding measurements, and determined crystal structures under the direction of K.D.C.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bing Ren.

Integrated supplementary information

  1. Supplementary Figure 1 The genomic distribution of candidate H3K4me1 binders correlates with enhancers in mESCs.

    a, ChIP–qPCR with H3K4me1, H3K4me3, and H3K27ac antibodies; primers were designed for previously validated mESC enhancers E147, E132, E110, E151, and E8 and for negative-control region N9. Data are shown as means ± s.d.; n = 3 biological replicates. b, Left, browser shot of the E110 enhancer region. Right, browser shot of the N9 negative-control region. c, Browser shot of CR binding to the Nanog enhancer region. The left box highlights the active enhancer, and the right box highlights the poised enhancer. d, Browser shot of CR binding to the mESC-specific miR290 super-enhancer. e, Bar plots showing the fraction of enhancers occupied by CRs for active enhancers (n = 13,811) versus poised enhancers (n = 28,008); related to Fig. 2d. ChIP–seq experiments were repeated at least twice with each antibody.

  2. Supplementary Figure 2 ChIP analysis of CR and histone modifications in WT and KMT2C/D DKO cells.

    a, Loss of CR binding at the miR290 super-enhancer in KMT2C/D DKO mESCs. b, KMT2C/D-dependent site. c, KMT2C/D-independent site. Each experiment was repeated at least twice. d, Top, pie chart showing the fraction of H3K4me2 peaks in KMT2C/D DKO mESCs according to KMT2C/D-dependent and KMT2C/D-independent patterns. Bottom, 2 × 2 table of the relationship with enhancer regions according to KMT2C/D-dependent and KMT2C/D-independent H3K4me2 peak regions. e, ChIP–qPCR analysis of the CRs listed at the miR290-295 super-enhancer, the Nanog ESC enhancer, and a normal enhancer. The fold difference in binding for WT versus DKO cells was calculated from the average of three replicates.

  3. Supplementary Figure 3 Depletion of H3K4me1 is associated with reduced binding of BAF components in KMT2C/D catalytically null (dCD) cells.

    a, Left, predicted domains of mouse DPF2 and DPF3. Right, corresponding aligned sequence with the PHD domains in green. b, Distribution of distal H3K4me1, H3K4me2, and H3K4me3 regions in dCD cells as compared to WT cells. c, Heat map of ChIP–seq signal for DPF2, H3K27ac, H3K4me2, and H3K4me3 at distal H3K4me1 regions. Regions are sorted by strength of H3K4me1 signal. d, Aggregate plots showing average DPF2, H3K27ac, H3K4me2, and H3K4me3 ChIP signal in WT and dCD cells over the same regions in c.

  4. Supplementary Figure 4 Purified BAF complex binding and remodeling activity.

    a, Silver staining of purified FLAG-BAF complex. Subunits and sizes are indicated on the left, and ladder is on the right. A representative gel is shown for four replicate preparations. b, FLAG-BAF binding to methylated peptides. Binding was assayed by western blotting with FLAG antibody; the assay was repeated twice. c, Polyacrylamide gels showing the n = 4 replicate assays of nucleosome remodeling quantified in Fig. 4c.

  5. Supplementary Figure 5 Binding of BAF45C to histone H3 tail peptides and structure of the BAF45C–H3 tail complex.

    a, Isothermal titration calorimetry data showing interaction of the H31–18 H4K4me0/H3K14ac peptide (injectant) with purified DPF3 PHD1–PHD2 region. The K value of 1.28 × 105 ± 7.38 × 103 M–1 corresponds to a Kd of 7.8 ± 0.5 μM. b, Binding to H31–18 H4K4me1/H3K14ac. The K value of 4.90 × 104 ± 5.48 × 103 M–1 corresponds to a Kd of 20.4 ± 2.3 μM. c, Binding to H31–18 H4K4me3/H3K14ac. The K value of 8.72 × 103 ± 5.63 × 103 M–1 corresponds to a Kd of 115 ± 128 μM. d, Experimental electron density calculated from a single-wavelength Zn SAD dataset. e, Refined 2FoFc electron density for the BAF45C–H3K4me0 complex. f, Refined 2FoFc electron density for the DPF3–H3K4me1 complex. The experiment was performed once.

  6. Supplementary Figure 6 Uncropped gel/western blot images.

    a, Corresponds to Fig. 1. b, Corresponds to Fig. 5. c, Corresponds to Supplementary Fig. 4.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–6.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Results from SILAC experiments.

  4. Supplementary tables 2–5

    Supplementary Tables 2–5.

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https://doi.org/10.1038/s41588-017-0015-6

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