Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Identification of H3K4me1-associated proteins at mammalian enhancers

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Identification of H3K4me1-binding proteins using SILAC and mass spectrometry analysis.
Fig. 2: Binding of chromatin regulators at H3K4me1-marked regions and enhancers.
Fig. 3: Concomitant loss of H3K4me1 and CR binding at enhancers in KMT2C/D DKO mESCs.
Fig. 4: Reduced BAF complex binding is associated with depletion of H3K4me1 in cells with catalytically null KMT2C/D.
Fig. 5: BAF complex preferentially binds to and remodels H3K4me1-modified nucleosomes.
Fig. 6: Structural basis for H3K4 recognition by DPF3.

References

  1. Hardison, R. C. & Taylor, J. Genomic approaches towards finding cis-regulatory modules in animals. Nat. Rev. Genet. 13, 469–483 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Heinz, S., Romanoski, C. E., Benner, C. & Glass, C. K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Taylor, G. C., Eskeland, R., Hekimoglu-Balkan, B., Pradeepa, M. M. & Bickmore, W. A. H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction. Genome Res. 23, 2053–2065 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Calo, E. & Wysocka, J. Modification of enhancer chromatin: what, how, and why? Mol. Cell 49, 825–837 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Musselman, C. A., Lalonde, M. E., Côté, J. & Kutateladze, T. G. Perceiving the epigenetic landscape through histone readers. Nat. Struct. Mol. Biol. 19, 1218–1227 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Seet, B. T., Dikic, I., Zhou, M. M. & Pawson, T. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7, 473–483 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Smith, E. & Shilatifard, A. The chromatin signaling pathway: diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes. Mol. Cell 40, 689–701 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58–69 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Hu, D. et al. The MLL3/MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers. Mol. Cell. Biol. 33, 4745–4754 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee, J. E. et al. H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. eLife 2, e01503 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wang, C. et al. Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition. Proc. Natl. Acad. Sci. USA 113, 11871–11876 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Outchkourov, N. S. et al. Balancing of histone H3K4 methylation states by the Kdm5c/SMCX histone demethylase modulates promoter and enhancer function. Cell Rep. 3, 1071–1079 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Cheng, J. et al. A role for H3K4 monomethylation in gene repression and partitioning of chromatin readers. Mol. Cell 53, 979–992 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vermeulen, M. et al. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell 142, 967–980 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Carruthers, L. M., Tse, C., Walker, K. P. III & Hansen, J. C. Assembly of defined nucleosomal and chromatin arrays from pure components. Methods Enzymol. 304, 19–35 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol. Biol. 119, 1–16 (1999).

    CAS  PubMed  Google Scholar 

  24. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Simon, M. D. et al. The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128, 1003–1012 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Engelen, E. et al. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry. Nat. Commun. 6, 7155 (2015).

    Article  PubMed  Google Scholar 

  27. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dreveny, I. et al. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification. Nucleic Acids Res 42, 822–835 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Yue, F. et al. A comparative encyclopedia of DNA elements in the mouse genome. Nature 515, 355–364 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li, Y. et al. CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells. PLoS One 9, e114485 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Dorighi, K. M. et al. Mll3 and Mll4 facilitate enhancer RNA synthesis and transcription from promoters independently of H3K4 monomethylation. Mol. Cell 66, 568–576 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Phelan, M. L., Sif, S., Narlikar, G. J. & Kingston, R. E. Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Mol. Cell 3, 247–253 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Zeng, L. et al. Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b. Nature 466, 258–262 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Li, W., Zhao, A., Tempel, W., Loppnau, P. & Liu, Y. Crystal structure of DPF3b in complex with an acetylated histone peptide. J. Struct. Biol. 195, 365–372 (2016).

    Article  CAS  PubMed  Google Scholar 

  35. Xiong, X. et al. Selective recognition of histone crotonylation by double PHD fingers of MOZ and DPF2. Nat. Chem. Biol. 12, 1111–1118 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ho, L. & Crabtree, G. R. Chromatin remodelling during development. Nature 463, 474–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rickels, R. et al. Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability. Nat. Genet. 49, 1647–1653 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Carey, M. F., Peterson, C. L. & Smale, S. T. Dignam and Roeder nuclear extract preparation. Cold Spring Harb. Protoc. 2009, pdb.prot5330 (2009).

    Article  PubMed  Google Scholar 

  40. Dignam, J. D., Martin, P. L., Shastry, B. S. & Roeder, R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 101, 582–598 (1983).

    Article  CAS  PubMed  Google Scholar 

  41. Kapust, R. B. & Waugh, D. S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Carey, M. F., Peterson, C. L. & Smale, S. T. In vivo DNase I, MNase, and restriction enzyme footprinting via ligation-mediated polymerase chain reaction (LM-PCR). Cold Spring Harb. Protoc. 2009, pdb.prot5277 (2009).

    Article  PubMed  Google Scholar 

  43. Albuquerque, C. P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol. Cell. Proteomics 7, 1389–1396 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Chen, S. H., Albuquerque, C. P., Liang, J., Suhandynata, R. T. & Zhou, H. A proteome-wide analysis of kinase–substrate network in the DNA damage response. J. Biol. Chem. 285, 12803–12812 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).

    Article  CAS  PubMed  Google Scholar 

  46. Cao, X. J., Arnaudo, A. M. & Garcia, B. A. Large-scale global identification of protein lysine methylation in vivo. Epigenetics 8, 477–485 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yun, M., Ruan, C., Huh, J. W. & Li, B. Reconstitution of modified chromatin templates for in vitro functional assays. Methods Mol. Biol. 833, 237–253 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kim, T. H. et al. Direct isolation and identification of promoters in the human genome. Genome Res. 15, 830–839 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Li, Z. et al. A global transcriptional regulatory role for c-Myc in Burkitt’s lymphoma cells. Proc. Natl. Acad. Sci. USA 100, 8164–8169 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Kabsch, W. Xds Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582–601 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. McCoy, A. J., Storoni, L. C. & Read, R. J. Simple algorithm for a maximum-likelihood SAD function. Acta Crystallogr. D Biol. Crystallogr. 60, 1220–1228 (2004).

    Article  PubMed  CAS  Google Scholar 

  56. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Terwilliger, T. C. Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D Biol. Crystallogr. 59, 38–44 (2003).

    Article  PubMed  CAS  Google Scholar 

  59. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Bing Ren.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

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.

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.

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.

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.

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.

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

Supplementary Text and Figures

Supplementary Figures 1–6.

Life Sciences Reporting Summary

Supplementary Table 1

Results from SILAC experiments.

Supplementary tables 2–5

Supplementary Tables 2–5.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Local, A., Huang, H., Albuquerque, C.P. et al. Identification of H3K4me1-associated proteins at mammalian enhancers. Nat Genet 50, 73–82 (2018). https://doi.org/10.1038/s41588-017-0015-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing