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Chromatin Immunoprecipitation Assays on Medulloblastoma Cell Line DAOY

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Medulloblastoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2423))

Abstract

Studies of DNA–protein interactions have revealed regulatory mechanisms of DNA replication, repair, remodeling, and transcription. Perturbation of any or all of these processes result in differential gene expression that can lead to tumor development. Chromatin immunoprecipitation assay (ChIP), currently the only method available to explore DNA-binding in vivo, has become a vastly utilized tool for cancer research. In this article we discuss an assay specified for a pediatric medulloblastoma (MB) cell line DAOY used to determine binding of transcription factors, to detect histone modifications, and to identify novel therapeutic targets.

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References

  1. Roussel MF, Stripay JL (2018) Epigenetic drivers in pediatric medulloblastoma. Cerebellum 17:28–36

    Article  CAS  PubMed  Google Scholar 

  2. Ahuja N, Sharma AR, Baylin SB (2016) Epigenetic therapeutics: a new weapon in the war against cancer. Annu Rev Med 67:73–89

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Meng X, Brodsky MH, Wolfe SA (2005) A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nat Biotechnol 23:988–994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Singh H, LeBowitz JH, Baldwin AS Jr, Sharp PA (1988) Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell 52:415–423

    Article  CAS  PubMed  Google Scholar 

  5. Galas DJ, Schmitz A (1978) DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5:3157–3170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fried MG (1989) Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10:366–376

    Article  CAS  PubMed  Google Scholar 

  7. Dedon PC, Soults JA, Allis CD, Gorovsky MA (1991) A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal Biochem 197:83–90

    Article  CAS  PubMed  Google Scholar 

  8. Biosmart (2015) Principle and protocol of chromatin immunoprecipitation (ChIP). Creative Biosmart Blog

    Google Scholar 

  9. Farnham PJ (2009) Insights from genomic profiling of transcription factors. Nat Rev Genet 10:605–616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dasgupta P, Chellappan SP (2007) Chromatin immunoprecipitation assays: molecular analysis of chromatin modification and gene regulation. Methods Mol Biol 383:135–152

    CAS  PubMed  Google Scholar 

  11. Gade P, Kalvakolanu DV (2012) Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity. Methods Mol Biol 809:85–104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Milne TA, Zhao K, Hess JL (2009) Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin-associated proteins. Methods Mol Biol 538:409–423

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pillai S, Dasgupta P, Chellappan SP (2009) Chromatin immunoprecipitation assays: analyzing transcription factor binding and histone modifications in vivo. Methods Mol Biol 523:323–339

    Article  CAS  PubMed  Google Scholar 

  14. Yan Y, Chen H, Costa M (2004) Chromatin immunoprecipitation assays. Methods Mol Biol 287:9–19

    CAS  PubMed  Google Scholar 

  15. Rhee HS, Pugh BF (2011) Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147:1408–1419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Serandour AA, Brown GD, Cohen JD, Carroll JS (2013) Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties. Genome Biol 14:R147

    Article  PubMed  PubMed Central  Google Scholar 

  17. Stormo GD, Zhao Y (2010) Determining the specificity of protein-DNA interactions. Nat Rev Genet 11:751–760

    Article  CAS  PubMed  Google Scholar 

  18. Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts BS, Mackiewicz M, Mendenhall EM, Myers RM (2015) CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. Genome Res 25:1581–1589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shah RN, Grzybowski AT, Cornett EM, Johnstone AL, Dickson BM, Boone BA, Cheek MA, Cowles MW, Maryanski D, Meiners MJ, Tiedemann RL, Vaughan RM, Arora N, Sun ZW, Rothbart SB, Keogh MC, Ruthenburg AJ (2018) Examining the roles of H3K4 methylation states with systematically characterized antibodies. Mol Cell 72:162–177.e7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mohammed H, Taylor C, Brown GD, Papachristou EK, Carroll JS, D'Santos CS (2016) Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc 11:316–326

    Article  CAS  PubMed  Google Scholar 

  21. Rafiee MR, Girardot C, Sigismondo G, Krijgsveld J (2016) Expanding the circuitry of pluripotency by selective isolation of chromatin-associated proteins. Mol Cell 64:624–635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang CI, Alekseyenko AA, LeRoy G, Elia AE, Gorchakov AA, Britton LM, Elledge SJ, Kharchenko PV, Garcia BA, Kuroda MI (2013) Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat Struct Mol Biol 20:202–209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dekker J, Rippe K, Dekker M, Kleckner N (2002) Capturing chromosome conformation. Science 295:1306–1311

    Article  CAS  PubMed  Google Scholar 

  24. Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, Orlov YL, Velkov S, Ho A, Mei PH, Chew EG, Huang PY, Welboren WJ, Han Y, Ooi HS, Ariyaratne PN, Vega VB, Luo Y, Tan PY, Choy PY, Wansa KD, Zhao B, Lim KS, Leow SC, Yow JS, Joseph R, Li H, Desai KV, Thomsen JS, Lee YK, Karuturi RK, Herve T, Bourque G, Stunnenberg HG, Ruan X, Cacheux-Rataboul V, Sung WK, Liu ET, Wei CL, Cheung E, Ruan Y (2009) An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 462:58–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fullwood MJ, Ruan Y (2009) ChIP-based methods for the identification of long-range chromatin interactions. J Cell Biochem 107:30–39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326:289–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY (2016) HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat Methods 13:919–922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Poorey K, Viswanathan R, Carver MN, Karpova TS, Cirimotich SM, McNally JG, Bekiranov S, Auble DT (2013) Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342:369–372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rotem A, Ram O, Shoresh N, Sperling RA, Goren A, Weitz DA, Bernstein BE (2015) Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol 33:1165–1172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dobson THW, Hatcher RJ, Swaminathan J, Das CM, Shaik S, Tao RH, Milite C, Castellano S, Taylor PH, Sbardella G, Gopalakrishnan V (2017) Regulation of USP37 expression by REST-associated G9a-dependent histone methylation. Mol Cancer Res 15:1073–1084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wright JB, Brown SJ, Cole MD (2010) Upregulation of c-MYC in cis through a large chromatin loop linked to a cancer risk-associated single-nucleotide polymorphism in colorectal cancer cells. Mol Cell Biol 30:1411–1420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mendez FM, Nunez FJ, Zorrilla-Veloz RI, Lowenstein PR, Castro MG (2018) Native chromatin immunoprecipitation using murine brain tumor neurospheres. J Vis Exp (131):57016

    Google Scholar 

  33. Benitez JA, Ma J, D'Antonio M, Boyer A, Camargo MF, Zanca C, Kelly S, Khodadadi-Jamayran A, Jameson NM, Andersen M, Miletic H, Saberi S, Frazer KA, Cavenee WK, Furnari FB (2017) PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3. Nat Commun 8:15223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bunt J, Hasselt NE, Zwijnenburg DA, Hamdi M, Koster J, Versteeg R, Kool M (2012) OTX2 directly activates cell cycle genes and inhibits differentiation in medulloblastoma cells. Int J Cancer 131:E21–E32

    Article  CAS  PubMed  Google Scholar 

  35. Shaked H, Shiff I, Kott-Gutkowski M, Siegfried Z, Haupt Y, Simon I (2008) Chromatin immunoprecipitation-on-chip reveals stress-dependent p53 occupancy in primary normal cells but not in established cell lines. Cancer Res 68:9671–9677

    Article  CAS  PubMed  Google Scholar 

  36. Northcott PA, Buchhalter I, Morrissy AS, Hovestadt V, Weischenfeldt J, Ehrenberger T, Grobner S, Segura-Wang M, Zichner T, Rudneva VA, Warnatz HJ, Sidiropoulos N, Phillips AH, Schumacher S, Kleinheinz K, Waszak SM, Erkek S, Jones DTW, Worst BC, Kool M, Zapatka M, Jager N, Chavez L, Hutter B, Bieg M, Paramasivam N, Heinold M, Gu Z, Ishaque N, Jager-Schmidt C, Imbusch CD, Jugold A, Hubschmann D, Risch T, Amstislavskiy V, Gonzalez FGR, Weber UD, Wolf S, Robinson GW, Zhou X, Wu G, Finkelstein D, Liu Y, Cavalli FMG, Luu B, Ramaswamy V, Wu X, Koster J, Ryzhova M, Cho YJ, Pomeroy SL, Herold-Mende C, Schuhmann M, Ebinger M, Liau LM, Mora J, McLendon RE, Jabado N, Kumabe T, Chuah E, Ma Y, Moore RA, Mungall AJ, Mungall KL, Thiessen N, Tse K, Wong T, Jones SJM, Witt O, Milde T, Von Deimling A, Capper D, Korshunov A, Yaspo ML, Kriwacki R, Gajjar A, Zhang J, Beroukhim R, Fraenkel E, Korbel JO, Brors B, Schlesner M, Eils R, Marra MA, Pfister SM, Taylor MD, Lichter P (2017) The whole-genome landscape of medulloblastoma subtypes. Nature 547:311–317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lin CY, Erkek S, Tong Y, Yin L, Federation AJ, Zapatka M, Haldipur P, Kawauchi D, Risch T, Warnatz HJ, Worst BC, Ju B, Orr BA, Zeid R, Polaski DR, Segura-Wang M, Waszak SM, Jones DT, Kool M, Hovestadt V, Buchhalter I, Sieber L, Johann P, Chavez L, Groschel S, Ryzhova M, Korshunov A, Chen W, Chizhikov VV, Millen KJ, Amstislavskiy V, Lehrach H, Yaspo ML, Eils R, Lichter P, Korbel JO, Pfister SM, Bradner JE, Northcott PA (2016) Active medulloblastoma enhancers reveal subgroup-specific cellular origins. Nature 530:57–62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hovestadt V, Jones DT, Picelli S, Wang W, Kool M, Northcott PA, Sultan M, Stachurski K, Ryzhova M, Warnatz HJ, Ralser M, Brun S, Bunt J, Jager N, Kleinheinz K, Erkek S, Weber UD, Bartholomae CC, von Kalle C, Lawerenz C, Eils J, Koster J, Versteeg R, Milde T, Witt O, Schmidt S, Wolf S, Pietsch T, Rutkowski S, Scheurlen W, Taylor MD, Brors B, Felsberg J, Reifenberger G, Borkhardt A, Lehrach H, Wechsler-Reya RJ, Eils R, Yaspo ML, Landgraf P, Korshunov A, Zapatka M, Radlwimmer B, Pfister SM, Lichter P (2014) Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing. Nature 510:537–541

    Article  CAS  PubMed  Google Scholar 

  39. Cejas P, Li L, O'Neill NK, Duarte M, Rao P, Bowden M, Zhou CW, Mendiola M, Burgos E, Feliu J, Moreno-Rubio J, Guadalajara H, Moreno V, Garcia-Olmo D, Bellmunt J, Mullane S, Hirsch M, Sweeney CJ, Richardson A, Liu XS, Brown M, Shivdasani RA, Long HW (2016) Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med 22:685–691

    Article  PubMed  Google Scholar 

  40. Barfeld SJ, Mills IG (2016) Mapping protein-DNA interactions using ChIP-exo and Illumina-based sequencing. Methods Mol Biol 1443:119–137

    Article  CAS  PubMed  Google Scholar 

  41. Wu M, Lin Z, Li X, Xin X, An J, Zheng Q, Yang Y, Lu D (2016) HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2. Sci Rep 6:36045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lin Z, Lu Y, Meng Q, Wang C, Li X, Yang Y, Xin X, Zheng Q, Xu J, Gui X, Li T, Pu H, Xiong W, Li J, Jia S, Lu D (2018) miR372 Promotes Progression of Liver Cancer Cells by Upregulating erbB-2 through Enhancement of YB-1. Mol Ther Nucleic Acids 11:494–507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schuijers J, Manteiga JC, Weintraub AS, Day DS, Zamudio AV, Hnisz D, Lee TI, Young RA (2018) Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism. Cell Rep 23:349–360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li G, Ruan X, Auerbach RK, Sandhu KS, Zheng M, Wang P, Poh HM, Goh Y, Lim J, Zhang J, Sim HS, Peh SQ, Mulawadi FH, Ong CT, Orlov YL, Hong S, Zhang Z, Landt S, Raha D, Euskirchen G, Wei CL, Ge W, Wang H, Davis C, Fisher-Aylor KI, Mortazavi A, Gerstein M, Gingeras T, Wold B, Sun Y, Fullwood MJ, Cheung E, Liu E, Sung WK, Snyder M, Ruan Y (2012) Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148:84–98

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Corces MR, Granja JM, Shams S, Louie BH, Seoane JA, Zhou W, Silva TC, Groeneveld C, Wong CK, Cho SW, Satpathy AT, Mumbach MR, Hoadley KA, Robertson AG, Sheffield NC, Felau I, Castro MAA, Berman BP, Staudt LM, Zenklusen JC, Laird PW, Curtis C, Cancer Genome Atlas Analysis Network, Greenleaf WJ, Chang HY (2018) The chromatin accessibility landscape of primary human cancers. Science 362:eaav1898

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wierer M, Mann M (2016) Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes. Hum Mol Genet 25:R106–R114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. D'Santos C, Taylor C, Carroll JS, Mohammed H (2015) RIME proteomics of estrogen and progesterone receptors in breast cancer. Data Brief 5:276–280

    Article  PubMed  PubMed Central  Google Scholar 

  48. Mohammed H, D'Santos C, Serandour AA, Ali HR, Brown GD, Atkins A, Rueda OM, Holmes KA, Theodorou V, Robinson JL, Zwart W, Saadi A, Ross-Innes CS, Chin SF, Menon S, Stingl J, Palmieri C, Caldas C, Carroll JS (2013) Endogenous purification reveals GREB1 as a key estrogen receptor regulatory factor. Cell Rep 3:342–349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mohammed H, Russell IA, Stark R, Rueda OM, Hickey TE, Tarulli GA, Serandour AA, Birrell SN, Bruna A, Saadi A, Menon S, Hadfield J, Pugh M, Raj GV, Brown GD, D'Santos C, Robinson JL, Silva G, Launchbury R, Perou CM, Stingl J, Caldas C, Tilley WD, Carroll JS (2015) Progesterone receptor modulates ERalpha action in breast cancer. Nature 523:313–317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Pediatrics, UT MD Anderson Cancer Center, Houston, TX, USA

    Tara Dobson & Jyothishmathi Swaminathan

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  1. Tara Dobson
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  2. Jyothishmathi Swaminathan
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Correspondence to Jyothishmathi Swaminathan .

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Editors and Affiliations

  1. Department of Pediatrics, Emory University, Atlanta, GA, USA

    Abhinav Dey

  2. Department of Pediatrics, Emory University, Atlanta, GA, USA

    Anshu Malhotra

  3. Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

    Neha Garg

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Dobson, T., Swaminathan, J. (2022). Chromatin Immunoprecipitation Assays on Medulloblastoma Cell Line DAOY. In: Dey, A., Malhotra, A., Garg, N. (eds) Medulloblastoma. Methods in Molecular Biology, vol 2423. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1952-0_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1952-0_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1951-3

  • Online ISBN: 978-1-0716-1952-0

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