Abstract
Post-translational modification of the histone proteins in chromatin plays a central role in the epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is, therefore, essential to understand the biochemical mechanisms that underlie genomic regulation. Here we present a synthetic biology method to engineer histones that bear site-specific modifications on cellular chromatin using protein trans-splicing (PTS). We genetically fused the N-terminal fragment of ultrafast split intein to the C terminus of histone H2B, which, on reaction with a complementary synthetic C intein, generated labelled histone. Using this approach, we incorporated various non-native chemical modifications into chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of PTS performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used PTS to semisynthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei and showed that this can trigger downstream epigenetic crosstalk of H3K79 methylation.
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References
Badeaux, A. I. & Shi, Y. Emerging roles for chromatin as a signal integration and storage platform. Nature Rev. Mol. Cell Biol. 14, 211–224 (2013).
Dawson, M. A. & Kouzarides, T. Cancer epigenetics: from mechanism to therapy. Cell 150, 12–27 (2012).
Sturm, D. et al. Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge. Nature Rev. Cancer 14, 92–107 (2014).
Rodriguez-Paredes, M. & Esteller, M. Cancer epigenetics reaches mainstream oncology. Nature Med. 17, 330–339 (2011).
Dunham, I. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
Allis, C. D. & Muir, T. W. Spreading chromatin into chemical biology. ChemBioChem 12, 264–279 (2011).
Fierz, B. & Muir, T. W. Chromatin as an expansive canvas for chemical biology. Nature Chem. Biol. 8, 417–427 (2012).
Jiang, H. et al. Regulation of transcription by the MLL2 complex and MLL complex-associated AKAP95. Nature Struct. Mol. Biol. 20, 1156–1163 (2013).
Ernst, J. & Kellis, M. Discovery and characterization of chromatin states for systematic annotation of the human genome. Nature Biotechnol. 28, 817–825 (2010).
Cole, P. A. Chemical probes for histone-modifying enzymes. Nature Chem. Biol. 4, 590–597 (2008).
Iwasaki, W. et al. Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains. Biochemistry 50, 7822–7832 (2011).
Shah, N. H., Dann, G. P., Vila-Perello, M., Liu, Z. & Muir, T. W. Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering. J. Am. Chem. Soc. 134, 11338–11341 (2012).
Wood, D. W. & Camarero, J. A. Intein applications: from protein purification and labeling to metabolic control methods. J. Biol. Chem. 289, 14512–14519 (2014).
Shah, N. H. & Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446–461 (2014).
Vila-Perello, M. et al. Streamlined expressed protein ligation using split inteins. J. Am. Chem. Soc. 135, 286–292 (2013).
Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT–HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature Med. 10, 310–315 (2004).
Giriat, I. & Muir, T. W. Protein semi-synthesis in living cells. J. Am. Chem. Soc. 125, 7180–7181 (2003).
Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nature Methods 7, 717–719 (2010).
Klein, T. et al. Live-cell dSTORM with SNAP-tag fusion proteins. Nature Methods 8, 7–9 (2011).
Pellois, J. P., Hahn, M. E. & Muir, T. W. Simultaneous triggering of protein activity and fluorescence. J. Am. Chem. Soc. 126, 7170–7171 (2004).
Borra, R., Dong, D., Elnagar, A. Y., Woldemariam, G. A. & Camarero, J. A. In-cell fluorescence activation and labeling of proteins mediated by FRET-quenched split inteins. J. Am. Chem. Soc. 134, 6344–6353 (2012).
Li, B., Carey, M. & Workman, J. L. The role of chromatin during transcription. Cell 128, 707–719 (2007).
Woodcock, C. L. & Ghosh, R. P. Chromatin higher-order structure and dynamics. Cold Spring Harb. Perspect. Biol. 2, a000596 (2010).
Salina, D. et al. Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108, 97–107 (2002).
Voigt, P. & Reinberg, D. Histone tails: ideal motifs for probing epigenetics through chemical biology approaches. ChemBioChem 12, 236–252 (2011).
Fuchs, G. & Oren, M. Writing and reading H2B monoubiquitylation. Biochim. Biophys. Acta 1839, 694–701 (2014).
Hodgins, R. R., Ellison, K. S. & Ellison, M. J. Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency. J. Biol. Chem. 267, 8807–8812 (1992).
McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G. & Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008).
McGinty, R. K. et al. Structure–activity analysis of semisynthetic nucleosomes: mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958–968 (2009).
Wilkins, B. J. et al. A cascade of histone modifications induces chromatin condensation in mitosis. Science 343, 77–80 (2014).
Matsushita, N. et al. A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair. Mol. Cell 19, 841–847 (2005).
Vlaming, H. et al. Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo. EMBO Rep. 15, 1220–1221 (2014).
Schwarze, S. R., Hruska, K. A. & Dowdy, S. F. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 10, 290–295 (2000).
van den Berg, A. & Dowdy, S. F. Protein transduction domain delivery of therapeutic macromolecules. Curr. Opin. Biotechnol. 22, 888–893 (2011).
Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nature Methods 11, 861–867 (2014).
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnol. 33, 73–80 (2015).
Musselman, C. A., Lalonde, M. E., Cote, J. & Kutateladze, T. G. Perceiving the epigenetic landscape through histone readers. Nature Struct. Mol. Biol. 19, 1218–1227 (2012).
Tan, M. et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 146, 1016–1028 (2011).
Tsompana, M. & Buck, M. J. Chromatin accessibility: a window into the genome. Epigenetics Chromatin 7, 33 (2014).
Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).
Crawford, G. E. et al. DNase-CHiP: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nature Methods 3, 503–509 (2006).
Crawford, G. E. et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 16, 123–131 (2006).
Kanda, T., Sullivan, K. F. & Wahl, G. M. Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385 (1998).
Sugita, T. et al. Improved cytosolic translocation and tumor-killing activity of Tat–shepherdin conjugates mediated by co-treatment with Tat-fused endosome-disruptive HA2 peptide. Biochem. Biophys. Res. Commun. 363, 1027–1032 (2007).
Goldberg, A. D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).
Acknowledgements
The authors thank the current and former members of the Muir laboratory for many valuable discussions. We further thank D. H. Perlman (Princeton Proteomics and Mass Spectrometry Core, Princeton University) for the mass spectrometry data and G. Laevsky (Confocal Core Facility, Princeton University) for help with the microscopy experiments. We also thank W. Wang, D. Storton and J. Miller (Microarray Facility, Princeton University) for help with the qPCR and RNA-seq experiments. Finally, we thank S. Josefowicz, C. Li and D. Allis (Rockefeller University) for help with the ChIP assays. This research was supported by the US National Institutes of Health (grants R37-GM086868 and R01 GM107047).
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Y.D., M.V-P. and T.W.M. conceived and designed the research. Y.D., M.V-P. and S.V. prepared the reagents and performed the experiments. Y.D. and T.W.M. wrote the manuscript.
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David, Y., Vila-Perelló, M., Verma, S. et al. Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nature Chem 7, 394–402 (2015). https://doi.org/10.1038/nchem.2224
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DOI: https://doi.org/10.1038/nchem.2224
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