Advertisement

Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging

  • Roubina Tatavosian
  • Xiaojun RenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1689)

Abstract

Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein–nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.

Key words

Epigenetics Epigenetic complex Chromatin Polycomb group (PcG) proteins PRC2 PRC1 Nucleosome Chromatin-binding mechanism Epigenetic protein–nucleosome complex Single-molecule fluorescent microscopy 

Notes

Acknowledgment

This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to X.R.).

References

  1. 1.
    Luger K, Dechassa ML, Tremethick DJ (2012) New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell Biol 13:436–447CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080CrossRefPubMedGoogle Scholar
  3. 3.
    Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. Cell 128:707–719CrossRefPubMedGoogle Scholar
  4. 4.
    Kerppola TK (2009) Polycomb group complexes—many combinations, many functions. Trends Cell Biol 19:692–704CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Simon JA, Kingston RE (2013) Occupying chromatin: polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 49:808–824CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Di Croce L, Helin K (2013) Transcriptional regulation by polycomb group proteins. Nat Struct Mol Biol 20:1147–1155CrossRefPubMedGoogle Scholar
  7. 7.
    Aranda S, Mas G, Di Croce L (2015) Regulation of gene transcription by polycomb proteins. Sci Adv 1:e1500737CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Blackledge NP, Rose NR, Klose RJ (2015) Targeting polycomb systems to regulate gene expression: modifications to a complex story. Nat Rev Mol Cell Biol 16:643–649CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kondo T, Ito S, Koseki H (2016) Polycomb in transcriptional phase transition of developmental genes. Trends Biochem Sci 41:9–19CrossRefPubMedGoogle Scholar
  10. 10.
    Margueron R, Reinberg D (2011) The polycomb complex PRC2 and its mark in life. Nature 469:343–349CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Steffen PA, Ringrose L (2014) What are memories made of? How polycomb and trithorax proteins mediate epigenetic memory. Nat Rev Mol Cell Biol 15:340–356CrossRefPubMedGoogle Scholar
  12. 12.
    Schwartz YB, Pirrotta V (2013) A new world of polycombs: unexpected partnerships and emerging functions. Nat Rev Genet 14:853–864CrossRefPubMedGoogle Scholar
  13. 13.
    Ren X, Vincenz C, Kerppola TK (2008) Changes in the distributions and dynamics of polycomb repressive complexes during embryonic stem cell differentiation. Mol Cell Biol 28:2884–2895CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ren X, Kerppola TK (2011) REST interacts with Cbx proteins and regulates polycomb repressive complex 1 occupancy at RE1 elements. Mol Cell Biol 31:2100–2110CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cheng B, Ren X, Kerppola TK (2014) KAP1 represses differentiation-inducible genes in embryonic stem cells through cooperative binding with PRC1 and derepresses pluripotency-associated genes. Mol Cell Biol 34:2075–2091CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhen CY, Duc HN, Kokotovic M, Phiel CJ, Ren X (2014) Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes. Mol Biol Cell 25:3726–3739CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tatavosian R, Zhen CY, Duc HN, Balas MM, Johnson AM, Ren X (2015) Distinct cellular assembly stoichiometry of polycomb complexes on chromatin revealed by single-molecule chromatin immunoprecipitation imaging. J Biol Chem 290:28038–28054CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhen CY, Tatavosian R, Huynh TN, Duc HN, Das R, Kokotovic M, Grimm JB, Lavis LD, Lee J, Mejia FJ, Li Y, Yao T, Ren X (2016) Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5. doi: 10.7554/eLife.17667
  19. 19.
    Voigt P, LeRoy G, Drury WJ 3rd, Zee BM, Son J, Beck DB, Young NL, Garcia BA, Reinberg D (2012) Asymmetrically modified nucleosomes. Cell 151:181–193CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Sadeh R, Launer-Wachs R, Wandel H, Rahat A, Friedman N (2016) Elucidating combinatorial chromatin states at single-nucleosome resolution. Mol Cell 63:1080–1088CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Jain A, Liu R, Ramani B, Arauz E, Ishitsuka Y, Ragunathan K, Park J, Chen J, Xiang YK, Ha T (2011) Probing cellular protein complexes using single-molecule pull-down. Nature 473:484–488CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ulbrich MH, Isacoff EY (2007) Subunit counting in membrane-bound proteins. Nat Methods 4:319–321PubMedPubMedCentralGoogle Scholar
  23. 23.
    Ren X, Li H, Clarke RW, Alves DA, Ying L, Klenerman D, Balasubramanian S (2006) Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy. J Am Chem Soc 128:4992–5000CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ren X, Gavory G, Li H, Ying L, Klenerman D, Balasubramanian S (2003) Identification of a new RNA.RNA interaction site for human telomerase RNA (hTR): structural implications for hTR accumulation and a dyskeratosis congenita point mutation. Nucleic Acids Res 31:6509–6515CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tatavosian R, Zhen CY, Ren XJ (2015) Single-molecule fluorescence microscopy methods in chromatin biology. ACS Symp Ser 1215:129–136CrossRefGoogle Scholar
  26. 26.
    Aggarwal V, Ha T (2016) Single-molecule fluorescence microscopy of native macromolecular complexes. Curr Opin Struct Biol 41:225–232CrossRefPubMedGoogle Scholar
  27. 27.
    Murphy PJ, Cipriany BR, Wallin CB, Ju CY, Szeto K, Hagarman JA, Benitez JJ, Craighead HG, Soloway PD (2013) Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells. Proc Natl Acad Sci U S A 110:7772–7777CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Shema E, Jones D, Shoresh N, Donohue L, Ram O, Bernstein BE (2016) Single-molecule decoding of combinatorially modified nucleosomes. Science 352:717–721CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Jain A, Liu R, Xiang YK, Ha T (2012) Single-molecule pull-down for studying protein interactions. Nat Protoc 7:445–452CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chung SH, Kennedy RA (1991) Forward-backward non-linear filtering technique for extracting small biological signals from noise. J Neurosci Methods 40:71–86CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Colorado DenverDenverUSA
  2. 2.Department of ChemistryUniversity of Colorado DenverDenverUSA

Personalised recommendations