Topoisomerase II Chromatin Immunoprecipitation

  • Kayleigh A. Smith
  • Ian G. Cowell
  • Caroline A. AustinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)


Chromatin immunoprecipitation is a method to isolate a protein of interest coupled to DNA following cross-linking with formaldehyde and to quantify the relative abundance or occupancy of the protein at specific genomic loci. After immunoprecipitation of protein–DNA complexes protein–DNA cross-links are reversed and the DNA is extracted. Various methods exist to identify binding sites and determine relative occupancy of the protein of interest; these include quantitative PCR, probing microarrays or sequencing the isolated DNA (ChIP-seq). This chapter details the method of chromatin immunoprecipitation of TOP2 to the point of DNA extraction from the precipitated protein–DNA complexes.

Key words

Topoisomerase II TOP2 Chromatin immunoprecipitation 


  1. 1.
    Lyu YL, Lin CP, Azarova AM, Cai L, Wang JC, Liu LF (2006) Role of topoisomerase IIbeta in the expression of developmentally regulated genes. Mol Cell Biol 26:7929–7941CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    BG J, Lunyak VV, Perissi V, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG (2006) A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription. Science 312:1798–1802CrossRefGoogle Scholar
  3. 3.
    Sano K, Miyaji-Yamaguchi M, Tsutsui KM, Tsutsui K (2008) Topoisomerase IIbeta activates a subset of neuronal genes that are repressed in AT-rich genomic environment. PLoS One 3(12):e4103. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Perillo B, Ombra MN, Bertoni A, Cuozzo C, Sacchetti S, Sasso A, Chiariotti L, Malorni A, Abbondanza C, Avvedimento EV (2008) DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science 319:202–206CrossRefPubMedGoogle Scholar
  5. 5.
    McNamara S, Wang H, Hanna N, Miller WH Jr (2008) Topoisomerase IIbeta negatively modulates retinoic acid receptor alpha function: a novel mechanism of retinoic acid resistance. Mol Cell Biol 28(6):2066–2077CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    McNamara S, Nichol JN, Wang H, Miller WH Jr (2010) Targeting PKC delta-mediated topoisomerase II beta overexpression subverts the differentiation block in a retinoic acid-resistant APL cell line. Leukemia 24(4):729–739CrossRefPubMedGoogle Scholar
  7. 7.
    Haffner MC, Aryee MJ, Toubaji A, Esopi DM, Albadine R, Gurel B, Isaacs WB, Bova GS, Liu W, Xu J, Meeker AK, Netto G, De Marzo AM, Nelson WG, Yegnasubramanian S (2010) Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nat Genet 42(8):668–675CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Tiwari VK, Burger L, Nikoletopoulou V, Deogracias R, Thakurela S, Wirbelauer C, Kaut J, Terranova R, Hoerner L, Mielke C, Boege F, Murr R, Peters AH, Barde YA, Schübeler D (2012) Target genes of topoisomerase IIβ regulate neuronal survival and are defined by their chromatin state. Proc Natl Acad Sci U S A 109(16):E934–E943CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Thakurela S, Garding A, Jung J, Schübeler D, Burger L, Tiwari VK (2013) Gene regulation and priming by topoisomerase IIα in embryonic stem cells. Nat Commun 4:2478CrossRefPubMedGoogle Scholar
  10. 10.
    Cowell IG, Sondka Z, Smith K, Lee KC, Manville CM, Sidorczuk-Lesthuruge M, Rance HA, Padget K, Jackson GH, Adachi N, Austin CA (2012) Model for MLL translocations in therapy-related leukemia involving topoisomerase IIβ-mediated DNA strand breaks and gene proximity. Proc Natl Acad Sci U S A 109(23):8989–8994CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Naughton C, Avlonitis N, Corless S, Prendergast JG, Mati IK, Eijk PP, Cockroft SL, Bradley M, Ylstra B, Gilbert N (2013) Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat Struct Mol Biol 20(3):387–395. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Smith KA, Cowell IG, Zhang Y, Sondka Z, Austin CA (2014) The role of topoisomerase II beta on breakage and proximity of RUNX1 to partner alleles RUNX1T1 and EVI1. Genes Chromosomes Cancer 53(2):117–128CrossRefPubMedGoogle Scholar
  13. 13.
    Zuchegna C, Aceto F, Bertoni A, Romano A, Perillo B, Laccetti P, Gottesman ME, Avvedimento EV, Porcellini A (2014) Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops. Nucleic Acids Res 42(17):11040–11055CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Madabhushi R, Gao F, Pfenning AR, Pan L, Yamakawa S, Seo J, Rueda R, Phan TX, Yamakawa H, Pao PC, Stott RT, Gjoneska E, Nott A, Cho S, Kellis M, Tsai LH (2015) Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell 161(7):1592–1605CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Manville CM, Smith K, Sondka Z, Rance H, Cockell S, Cowell IG, Lee KC, Morris NJ, Padget K, Jackson GH, Austin CA (2015) Genome-wide ChIP-seq analysis of human TOP2B occupancy in MCF7 breast cancer epithelial cells. Biol Open 4(11):1436–1447CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Uusküla-Reimand L, Hou H, Samavarchi-Tehrani P, Rudan MV, Liang M, Medina-Rivera A, Mohammed H, Schmidt D, Schwalie P, Young EJ, Reimand J, Hadjur S, Gingras AC, Wilson MD (2016) Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders. Genome Biol 17(1):182PubMedPubMedCentralGoogle Scholar
  17. 17.
    Landt SG, Marinov GK, Kundaje A, Kheradpour P, Pauli F, Batzoglou S, Bernstein BE, Bickel P, Brown JB, Cayting P, Chen Y, DeSalvo G, Epstein C, Fisher-Aylor KI, Euskirchen G, Gerstein M, Gertz J, Hartemink AJ, Hoffman MM, Iyer VR, Jung YL, Karmakar S, Kellis M, Kharchenko PV, Li Q, Liu T, Liu XS, Ma L, Milosavljevic A, Myers RM, Park PJ, Pazin MJ, Perry MD, Raha D, Reddy TE, Rozowsky J, Shoresh N, Sidow A, Slattery M, Stamatoyannopoulos JA, Tolstorukov MY, White KP, Xi S, Farnham PJ, Lieb JD, Wold BJ, Snyder M (2012) ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22(9):1813–1831CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kidder BL, Hu G, Zhao K (2011) ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol 12(10):918–922CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Kayleigh A. Smith
    • 1
  • Ian G. Cowell
    • 1
  • Caroline A. Austin
    • 1
    Email author
  1. 1.Institute for Cell and Molecular BiosciencesUniversity of Newcastle upon TyneNewcastle upon TyneUK

Personalised recommendations