Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All sequencing datasets produced in this study have been deposited in the NCBI Gene Expression Omnibus under accession number GSE128907. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014483 and PXD017235. Additional genomics datasets used in this study include U2OS FLASH77 (GSE69149); U2OS H3K4me3 (ref. 78) (GSE87831); mESC whole-genome bisulfite sequencing79 (GSE30206); mESC TAF3 (ref. 80) (GSE30959); mESC CBX7 ref. 81) (GSE42466); mESC CBX1 (ref. 27) (GSE71114); mESC bio-tagged MBD1 (ref. 22) (GSE39610); and mESC H4K20me3 from ENCODE (SRR094944).
Allis, C. D. & Jenuwein, T. The molecular hallmarks of epigenetic control. Nat. Rev. Genet. 17, 487–500 (2016).
Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. & Patel, D. J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).
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).
Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).
Vermeulen, M. et al. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell 142, 967–980 (2010).
Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).
Nikolov, M. et al. Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns. Mol. Cell. Proteomics 10, M110.005371 (2011).
Eberl, H. C., Spruijt, C. G., Kelstrup, C. D., Vermeulen, M. & Mann, M. A map of general and specialized chromatin readers in mouse tissues generated by label-free interaction proteomics. Mol. Cell 49, 368–378 (2013).
Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).
Mittler, G., Butter, F. & Mann, M. A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements. Genome Res. 19, 284–293 (2008).
Déjardin, J. & Kingston, R. E. Purification of proteins associated with specific genomic loci. Cell 136, 175–186 (2009).
Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043 (2017).
Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 437–439 (2018).
Schmidtmann, E., Anton, T., Rombaut, P., Herzog, F. & Leonhardt, H. Determination of local chromatin composition by CasID. Nucleus 7, 476–484 (2016).
Fischle, W. et al. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 17, 1870–1881 (2003).
Bernstein, E. et al. Mouse Polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin. Mol. Cell. Biol. 26, 2560–2569 (2006).
Bannister, A. J. et al. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120–124 (2001).
Lachner, M., O’Carroll, D., Rea, S., Mechtler, K. & Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116–120 (2001).
Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58–69 (2007).
Meehan, R. R., Lewis, J. D. & Bird, A. P. Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. Nucleic Acids Res. 20, 5085–5092 (1992).
Nan, X., Meehan, R. R. & Bird, A. Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Res. 21, 4886–4892 (1993).
Baubec, T., Ivanek, R., Lienert, F. & Schübeler, D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell 153, 480–492 (2013).
Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M. & Helin, K. The Polycomb group protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell. Biol. 27, 3769–3779 (2007).
Tsumura, A. et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006).
Peters, A. H. F. M. et al. Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nat. Genet. 30, 77–80 (2001).
Xu, J. et al. Super-resolution imaging of higher-order chromatin structures at different epigenomic states in single mammalian cells. Cell Rep. 24, 873–882 (2018).
Hiragami-Hamada, K. et al. Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin. Nat. Commun. 7, 1–16 (2016).
Yap, K. L. et al. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by Polycomb CBX7 in transcriptional silencing of INK4a. Mol. Cell 38, 662–674 (2010).
Jørgensen, H. F., Ben-Porath, I. & Bird, A. P. Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains. Mol. Cell. Biol. 24, 3387–3395 (2004).
Brown, K. et al. The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome. Hum. Mol. Genet. 25, 558–570 (2016).
Feller, C., Forné, I., Imhof, A. & Becker, P. B. Global and specific responses of the histone acetylome to systematic perturbation. Mol. Cell 57, 559–571 (2015).
Schotta, G. et al. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251–1262 (2004).
Fischle, W. et al. Regulation of HP1–chromatin binding by histone H3 methylation and phosphorylation. Nat. Cell Biol. 438, 1116–1122 (2005).
Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).
Kim, D. I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27, 1188–1196 (2016).
Ramanathan, M. et al. RNA–protein interaction detection in living cells. Nat. Meth. 15, 207–212 (2018).
Saksouk, N. et al. Redundant mechanisms to form silent chromatin at pericentromeric regions rely on BEND3 and DNA methylation. Mol. Cell 56, 580–594 (2014).
Tachibana, M. et al. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779–1791 (2002).
Peters, A. H. et al. Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. Mol. Cell 12, 1577–1589 (2003).
Tchasovnikarova, I. A. et al. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science 348, 1481–1485 (2015).
Goldberg, A. D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).
Agarwal, N. et al. MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. Nucleic Acids Res. 35, 5402–5408 (2007).
Arita, K. et al. Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1. Proc. Natl Acad. Sci. USA 109, 12950–12955 (2012).
Ueda, J., Tachibana, M., Ikura, T. & Shinkai, Y. Zinc finger protein Wiz links G9a/GLP histone methyltransferases to the co-repressor molecule CtBP. J. Biol. Chem. 281, 20120–20128 (2006).
Nozawa, R.-S. et al. Human POGZ modulates dissociation of HP1α from mitotic chromosome arms through Aurora B activation. Nat. Cell Biol. 12, 719–727 (2010).
Yin, Y. et al. Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science 356, eaaj2239-17 (2017).
Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).
Voigt, P. et al. Asymmetrically modified nucleosomes. Cell 151, 181–193 (2012).
Yang, X.-C., Burch, B. D., Yan, Y., Marzluff, W. F. & Dominski, Z. FLASH, a proapoptotic protein involved in activation of caspase-8, is essential for 3′ end processing of histone pre-mRNAs. Mol. Cell 36, 267–278 (2009).
Doyon, Y., Selleck, W., Lane, W. S., Tan, S. & Côté, J. Structural and functional conservation of the Nua4 histone acetyltransferase complex from yeast to humans. Mol. Cell. Biol. 24, 1884–1896 (2004).
Ravens, S., Yu, C., Ye, T., Stierle, M. & Tora, L. Tip60 complex binds to active Pol II promoters and a subset of enhancers and co-regulates the c-Myc network in mouse embryonic stem cells. Epigenetics Chromatin 8, 45 (2015).
Whetstine, J. R. et al. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125, 467–481 (2006).
Das, P. P. et al. Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cell identity. Mol. Cell 53, 32–48 (2014).
Horton, J. R. et al. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases. Nat. Struct. Mol. Biol. 17, 38–43 (2010).
Aranda, S., Mas, G. & Di Croce, L. Regulation of gene transcription by Polycomb proteins. Sci. Adv. 1, e1500737 (2015).
Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).
Delachat, A. M. F. et al. Engineered multivalent sensors to detect coexisting histone modifications in living stem cells. Cell Chem. Biol. 25, 51–56 (2018).
Mauser, R., Kungulovski, G., Keup, C., Reinhardt, R. & Jeltsch, A. Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state. Epigenetics Chromatin 10, 45 (2017).
Tekel, S. J. et al. Tandem histone-binding domains enhance the activity of a synthetic chromatin effector. ACS Synth. Biol. 7, 842–852 (2018).
Flemr, M. & Bühler, M. Single-step generation of conditional knockout mouse embryonic stem cells. Cell Rep. 12, 709–716 (2015).
Bibel, M., Richter, J., Lacroix, E. & Barde, Y.-A. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat. Protoc. 2, 1034–1043 (2007).
Abad, M. A. et al. Borealin–nucleosome interaction secures chromosome association of the chromosomal passenger complex. J. Cell Biol. 218, 3912–3925 (2019).
Manzo, M. et al. Isoform-specific localization of DNMT3A regulates DNA methylation fidelity at bivalent CpG islands. EMBO J. 36, 3421–3434 (2017).
Gaidatzis, D., Lerch, A., Hahne, F. & Stadler, M. B. QuasR: quantification and annotation of short reads in R. Bioinformatics 31, 1130–1132 (2015).
Derrien, T. et al. Fast computation and applications of genome mappability. PLoS One 7, e30377 (2012).
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).
Ernst, J. & Kellis, M. Chromatin-state discovery and genome annotation with ChromHMM. Nat. Protoc. 12, 2478–2492 (2017).
Akalin, A., Franke, V., Vlahoviček, K., Mason, C. E. & Schübeler, D. Genomation: a toolkit to summarize, annotate and visualize genomic intervals. Bioinformatics 31, 1127–1129 (2014).
Feller, C. et al. Histone epiproteomic profiling distinguishes oligodendroglioma, IDH-mutant and 1p/19q co-deleted from IDH-mutant astrocytoma and reveals less tri-methylation of H3K27 in oligodendrogliomas. Acta Neuropathol. 139, 211–213 (2019).
Barkow-Oesterreicher, S., Türker, C. & Panse, C. FCC—an automated rule-based processing tool for life science data. Source Code Biol. Med. 8, 3 (2013).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).
Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).
Tsou, C.-C. et al. DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics. Nat. Methods 12, 258–264 (2015).
Szklarczyk, D. et al. STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 (2019).
Sokolova, M. et al. Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion. Cell Cycle 16, 189–199 (2017).
Ibarra, A., Benner, C., Tyagi, S., Cool, J. & Hetzer, M. W. Nucleoporin-mediated regulation of cell identity genes. Genes Dev. 30, 2253–2258 (2016).
Stadler, M. B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).
Liu, Z., Scannell, D. R., Eisen, M. B. & Tjian, R. Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146, 720–731 (2011).
Morey, L., Aloia, L., Cozzuto, L., Benitah, S. A. & Di Croce, L. RYBP and Cbx7 define specific biological functions of Polycomb complexes in mouse embryonic stem cells. Cell Rep. 3, 60–69 (2013).
We thank D. Schübeler (FMI, Basel) for providing the Dnmt-TKO embryonic stem cell line and P. A. Khavari (Stanford) for cDNA encoding the BASU biotin ligase. Furthermore, we thank B. Roschitzki, J. Grossmann, T. Kockmann, P. Knobel, M. Majchrzak and R. Klemm for initial discussions on biotin-ID methods and MS detection. We would like to thank members of the Functional Genomics Center Zurich for high-throughput sequencing and MS support, members of the Centre for Microscopy and Image Analysis for their support and the Science IT team at the University of Zurich for providing the computational infrastructure. We are grateful to the Edinburgh Protein Production Facility for their support. We thank V. Major for help with cloning and K. Webb for help with histone purification. Furthermore, we thank M. Altmeyer, A. Krebs, D. Schübeler and members of the Baubec laboratory for their critical input on the manuscript. The authors would like to acknowledge the following support: Swiss National Science Foundation 157488 and 180345 to T.B. and 107679 to R.A.; Swiss Initiative in Systems Biology (SystemsX.ch) 2015/322 to T.B.; ERC-AdvGr 670821-Proteomics 4D to R.A.; ERC-STG 639253 to P.V.; Innovative Medicines Initiative (EU/EFPIA) ULTRA-DD grant 115766 to R.A; Wellcome Trust (104175/Z/14/Z, Sir Henry Dale Fellowship) to P.V.; EMBO long-term fellowships to N.S. and C.F.; a UZH Forschungskredit fellowship to N.S.; a UZH Candoc fellowship to C.A.; Wellcome Trust Doctoral Studentship 105244 to E.B.; and Wellcome Trust core funding 203149 and 101527/Z/13/Z to Wellcome Centre for Cell Biology and Edinburgh Protein Production Facility, respectively.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–15
Dentified proximal interactome at H3K4me3, H3K27me3, H3K9me3 and bivalently marked H3K4me3/H3K27me3 sites. Identified proteins from ChromID experiments using histone modification eCRs, including log2FC, P values and a Boolean indicator if called significant.
Identified proximal interactome at 5-methyl-CpG DNA methylated sites. Identified proteins from ChromID experiments using the 5-methyl-CpG-specific eCR, including log2FC, P values and a Boolean indicator if called significant.
Estimation of protein abundance by data-independent acquisition in eCR-expressing cells. Protein abundance is based on average intensities calculated from the top three peptides per protein.
Time-lapse fluorescence imaging of proliferating eGFP+ control cells. Cells undergoing division at one selected region were imaged at 5‐min intervals for approximately 12.5 h. The video was generated using FIJI (version 2.0.0) and the Bio-Formats Importer plugin. An appropriate xy region and a single z plane were selected for further image series analysis and display. Scale bar, 5 µm.
Time-lapse fluorescence imaging of proliferating H3K27me3-reader cells. Cells undergoing division at one selected region were imaged at 5‐min intervals for approximately 12.5 h. The video was generated using FIJI (version 2.0.0) and the Bio-Formats Importer plugin. An appropriate xy region and a single z plane were selected for further image series analysis and display. Scale bar, 5 µm.
About this article
Cite this article
Villaseñor, R., Pfaendler, R., Ambrosi, C. et al. ChromID identifies the protein interactome at chromatin marks. Nat Biotechnol 38, 728–736 (2020). https://doi.org/10.1038/s41587-020-0434-2
Nature Communications (2021)
Dual DNA and protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia
Nature Methods (2021)
Nature Methods (2021)
Nature Chemistry (2020)
Nature Methods (2020)