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time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance

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Histone Variants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1832))

Abstract

Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.

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References

  1. Campos EI, Reinberg D (2009) Histones: annotating chromatin. Annu Rev Genet 43:559–599. https://doi.org/10.1146/annurev.genet.032608.103928

    Article  PubMed  CAS  Google Scholar 

  2. Ragunathan K, Jih G, Moazed D (2015) Epigenetics. Epigenetic inheritance uncoupled from sequence-specific recruitment. Science 348:1258699. https://doi.org/10.1126/science.1258699

    Article  PubMed  CAS  Google Scholar 

  3. Audergon PNCB, Catania S, Kagansky A et al (2015) Restricted epigenetic inheritance of H3K9 methylation. Science 348:132–135. https://doi.org/10.1126/science.1260638

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Hansen KH, Bracken AP, Pasini D et al (2008) A model for transmission of the H3K27me3 epigenetic mark. Nat Cell Biol 10:1291–1300. https://doi.org/10.1038/ncb1787

    Article  PubMed  CAS  Google Scholar 

  5. Margueron R, Justin N, Ohno K et al (2009) Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461:762–767. https://doi.org/10.1038/nature08398

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Berry S, Hartley M, Olsson TSG et al (2015) Local chromatin environment of a Polycomb target gene instructs its own epigenetic inheritance. elife 4:e07205. https://doi.org/10.7554/eLife.07205

    Article  PubMed Central  CAS  Google Scholar 

  7. Dodd IB, Micheelsen MA, Sneppen K, Thon G (2007) Theoretical analysis of epigenetic cell memory by nucleosome modification. Cell 129:813–822. https://doi.org/10.1016/j.cell.2007.02.053

    Article  PubMed  CAS  Google Scholar 

  8. Alabert C, Barth TK, Reverón-Gómez N et al (2015) Two distinct modes for propagation of histone PTMs across the cell cycle. Genes Dev 29:585–590. https://doi.org/10.1101/gad.256354.114

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Akiyama T, Suzuki O, Matsuda J, Aoki F (2011) Dynamic replacement of histone H3 variants reprograms epigenetic marks in early mouse embryos. PLoS Genet 7:e1002279. https://doi.org/10.1371/journal.pgen.1002279

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ng RK, Gurdon JB (2008) Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription. Nat Cell Biol 10:102–109. https://doi.org/10.1038/ncb1674

    Article  PubMed  CAS  Google Scholar 

  11. Jackson V (1990) In vivo studies on the dynamics of histone-DNA interaction: evidence for nucleosome dissolution during replication and transcription and a low level of dissolution independent of both. Biochemistry 29:719–731. https://doi.org/10.1021/bi00455a019

    Article  PubMed  CAS  Google Scholar 

  12. Xu M, Long C, Chen X et al (2010) Partitioning of histone H3-H4 tetramers during DNA replication–dependent chromatin assembly. Science 328:94–98. https://doi.org/10.1126/science.1178994

    Article  PubMed  CAS  Google Scholar 

  13. Alabert C, Bukowski-Wills J-C, Lee S-B et al (2014) Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 16:281–293. https://doi.org/10.1038/ncb2918

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Ahmad K, Henikoff S (2001) Centromeres are specialized replication domains in heterochromatin. J Cell Biol 153:101–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ahmad K, Henikoff S (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol Cell 9:1191–1200. https://doi.org/10.1016/S1097-2765(02)00542-7

    Article  PubMed  CAS  Google Scholar 

  16. Tran V, Lim C, Xie J, Chen X (2012) Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution. Science 338:679–682. https://doi.org/10.1126/science.1226028

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Kimura H, Cook PR (2001) Kinetics of Core histones in living human cells little exchange of H3 and H4 and some rapid exchange of H2b. J Cell Biol 153:1341–1354. https://doi.org/10.1083/jcb.153.7.1341

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Falk SJ, Guo LY, Sekulic N et al (2015) CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. Science 348:699–703. https://doi.org/10.1126/science.1259308

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Liu J, Vidi P-A, Lelievre SA, Irudayaraj JMK (2015) Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage. J Cell Sci 128:599–604. https://doi.org/10.1242/jcs.161885

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Damoiseaux R, Keppler A, Johnsson K (2001) Synthesis and applications of chemical probes for human O6-alkylguanine-DNA alkyltransferase. Chembiochem A Eur J Chem Biol 2:285–287

    Article  CAS  Google Scholar 

  21. Keppler A, Pick H, Arrivoli C et al (2004) Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101:9955–9959. https://doi.org/10.1073/pnas.0401923101

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Keppler A, Gendreizig S, Gronemeyer T et al (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89. https://doi.org/10.1038/nbt765

    Article  PubMed  CAS  Google Scholar 

  23. Jansen LET, Black BE, Foltz DR, Cleveland DW (2007) Propagation of centromeric chromatin requires exit from mitosis. J Cell Biol 176:795–805. https://doi.org/10.1083/jcb.200701066

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Silva MCC, Bodor DL, Stellfox ME et al (2012) Cdk activity couples epigenetic centromere inheritance to cell cycle progression. Dev Cell 22:52–63. https://doi.org/10.1016/j.devcel.2011.10.014

    Article  PubMed  CAS  Google Scholar 

  25. Carroll CW, Silva MCC, Godek KM et al (2009) Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nat Cell Biol 11:896–902. https://doi.org/10.1038/ncb1899

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Stankovic A, Guo LY, Mata JF et al (2017) A dual inhibitory mechanism sufficient to maintain cell-cycle-restricted CENP-A assembly. Mol Cell 65:231–246. https://doi.org/10.1016/j.molcel.2016.11.021

    Article  PubMed  CAS  Google Scholar 

  27. Bergmann JH, Rodríguez MG, Martins NMC et al (2011) Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. EMBO J 30:328–340. https://doi.org/10.1038/emboj.2010.329

    Article  PubMed  CAS  Google Scholar 

  28. Ray-Gallet D, Woolfe A, Vassias I et al (2011) Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol Cell 44:928–941. https://doi.org/10.1016/j.molcel.2011.12.006

    Article  PubMed  CAS  Google Scholar 

  29. Bodor DL, Rodríguez MG, Moreno N, Jansen LET (2012) Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging. Curr Protoc Cell Biol Chapter 8:Unit8.8. https://doi.org/10.1002/0471143030.cb0808s55

    Article  Google Scholar 

  30. Bodor DL, Valente LP, Mata JF et al (2013) Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes. Mol Biol Cell 24:923–932. https://doi.org/10.1091/mbc.E13-01-0034

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Dion MF, Kaplan T, Kim M et al (2007) Dynamics of replication-independent histone turnover in budding yeast. Science 315:1405–1408. https://doi.org/10.1126/science.1134053

    Article  PubMed  CAS  Google Scholar 

  32. Huang C, Zhang Z, Xu M et al (2013) H3.3-H4 tetramer splitting events feature cell-type specific enhancers. PLoS Genet 9:e1003558. https://doi.org/10.1371/journal.pgen.1003558

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Katan-Khaykovich Y, Struhl K (2011) Splitting of H3-H4 tetramers at transcriptionally active genes undergoing dynamic histone exchange. Proc Natl Acad Sci 108:1296–1301. https://doi.org/10.1073/pnas.1018308108

    Article  PubMed  Google Scholar 

  34. Deal RB, Henikoff JG, Henikoff S (2010) Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328:1161–1164. https://doi.org/10.1126/science.1186777

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Mito Y, Henikoff JG, Henikoff S (2005) Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 37:1090–1097. https://doi.org/10.1038/ng1637

    Article  PubMed  CAS  Google Scholar 

  36. Mito Y, Henikoff JG, Henikoff S (2007) Histone replacement marks the boundaries of cis-regulatory domains. Science 315:1408–1411. https://doi.org/10.1126/science.1134004

    Article  PubMed  CAS  Google Scholar 

  37. Verzijlbergen KFKF, Menendez-Benito V, Van Welsem T et al (2010) Recombination-induced tag exchange to track old and new proteins. Proc Natl Acad Sci 107:64–68. https://doi.org/10.1073/pnas.0911164107

    Article  PubMed  Google Scholar 

  38. Radman-Livaja M, Verzijlbergen KF, Weiner A et al (2011) Patterns and mechanisms of ancestral histone protein inheritance in budding yeast. PLoS Biol 9:e1001075. https://doi.org/10.1371/journal.pbio.1001075

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Corrêa IR, Baker B, Zhang A et al (2013) Substrates for improved live-cell fluorescence labeling of SNAP-tag. Curr Pharm Des 19:5414–5420

    Article  CAS  PubMed  Google Scholar 

  40. Sun X, Zhang A, Baker B et al (2011) Development of SNAP-tag Fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12:2217–2226. https://doi.org/10.1002/cbic.201100173

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Corrêa IR (2015) Considerations and protocols for the synthesis of custom protein labeling probes. In: Site-specific protein labeling, Methods and protocols. Humana Press, New York, pp 55–79

    Google Scholar 

  42. Jasencakova Z, Scharf AND, Ask K et al (2010) Replication stress interferes with histone recycling and predeposition marking of new histones. Mol Cell 37:736–743. https://doi.org/10.1016/j.molcel.2010.01.033

    Article  PubMed  CAS  Google Scholar 

  43. Deaton AM, Gómez-Rodríguez M, Mieczkowski J et al (2016) Enhancer regions show high histone H3.3 turnover that changes during differentiation. elife 5:e15316. https://doi.org/10.7554/eLife.15316

    Article  PubMed  PubMed Central  Google Scholar 

  44. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetjournal 17:10. https://doi.org/10.14806/ej.17.1.200

    Article  Google Scholar 

  45. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. https://doi.org/10.1093/bioinformatics/btp352

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Ramirez F, Dundar F, Diehl S et al (2014) deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res 42:W187–W191. https://doi.org/10.1093/nar/gku365

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Dunham I et al (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74. https://doi.org/10.1038/nature11247

    Article  CAS  Google Scholar 

  49. Robinson JT, Thorvaldsdóttir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26. https://doi.org/10.1038/nbt.1754

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgments

We thank João Mata (IGC) for technical support, Brenda Baker (New England Biolabs) for assistance with synthesis of CP-Biotin. Bianka Baying and Vladimir Benes (EMBL genecore) for NGS support. We are grateful to Alekos Athanasiadis (IGC) and EpiLab members for helpful comments on the manuscript.

This work is supported by Fundação para a Ciência e a Tecnologia (FCT) postdoctoral fellowship SFRH/BPD/117179/2016 (to WS) and doctoral fellowship SFRH/BD/33567/2008 (to MRG). Salary support to LETJ is provided by an “Investigador FCT” grant. Further support was provided by FCT grants BIA-BCM/100557/2008 and BIA-PRO/100537/2008, European Commission FP7 Program, Marie Curie Reintegration grant, an EMBO installation grant and an ERC consolidator grant ERC-2013-CoG-615638 to LETJ.

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Author notes

  1. Wojciech Siwek and Mariluz Gómez-Rodríguez contributed equally to this work.

Authors and Affiliations

  1. Instituto Gulbenkian de Ciência, Oeiras, Portugal

    Wojciech Siwek, Mariluz Gómez-Rodríguez, Daniel Sobral & Lars E. T. Jansen

  2. Departamento de Ciencias Naturales and Matemáticas, Pontificia Universidad Javeriana, Cali, Colombia

    Mariluz Gómez-Rodríguez

  3. New England Biolabs, Ipswich, MA, USA

    Ivan R. Corrêa Jr

Authors
  1. Wojciech Siwek
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  2. Mariluz Gómez-Rodríguez
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  3. Daniel Sobral
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  4. Ivan R. Corrêa Jr
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  5. Lars E. T. Jansen
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Corresponding author

Correspondence to Lars E. T. Jansen .

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Editors and Affiliations

  1. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Guillermo A. Orsi

  2. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Geneviève Almouzni

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Siwek, W., Gómez-Rodríguez, M., Sobral, D., Corrêa, I.R., Jansen, L.E.T. (2018). time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance. In: Orsi, G., Almouzni, G. (eds) Histone Variants. Methods in Molecular Biology, vol 1832. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8663-7_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8663-7_7

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  • Publisher Name: Humana Press, New York, NY

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