Acta Neuropathologica

, Volume 125, Issue 3, pp 385–394 | Cite as

OTX2 sustains a bivalent-like state of OTX2-bound promoters in medulloblastoma by maintaining their H3K27me3 levels

  • Jens Bunt
  • Nancy A. Hasselt
  • Danny A. Zwijnenburg
  • Jan Koster
  • Rogier Versteeg
  • Marcel Kool
Original Paper


Recent studies showed frequent mutations in histone H3 lysine 27 (H3K27) demethylases in medulloblastomas of Group 3 and Group 4, suggesting a role for H3K27 methylation in these tumors. Indeed, trimethylated H3K27 (H3K27me3) levels were shown to be higher in Group 3 and 4 tumors compared to WNT and SHH medulloblastomas, also in tumors without detectable mutations in demethylases. Here, we report that polycomb genes, required for H3K27 methylation, are consistently upregulated in Group 3 and 4 tumors. These tumors show high expression of the homeobox transcription factor OTX2. Silencing of OTX2 in D425 medulloblastoma cells resulted in downregulation of polycomb genes such as EZH2, EED, SUZ12 and RBBP4 and upregulation of H3K27 demethylases KDM6A, KDM6B, JARID2 and KDM7A. This was accompanied by decreased H3K27me3 and increased H3K27me1 levels in promoter regions. Strikingly, the decrease of H3K27me3 was most prominent in promoters that bind OTX2. OTX2-bound promoters showed high levels of the H3K4me3 and H3K9ac activation marks and intermediate levels of the H3K27me3 inactivation mark, reminiscent of a bivalent modification. After silencing of OTX2, H3K27me3 levels strongly dropped, but H3K4me3 and H3K9ac levels remained high. OTX2-bound bivalent genes showed high expression levels in D425, but the expression of most of these genes did not change after OTX2 silencing and loss of the H3K27me3 mark. Maintaining promoters in a bivalent state by sustaining H3K27 trimethylation therefore seems to be an important function of OTX2 in medulloblastoma, while other transcription factors might regulate the actual expression levels of these genes.


OTX2 Medulloblastoma Histone modification H3K27me3 Chromatin immunoprecipitation 



This research was supported by the Tom Voûte Fonds. The authors thank M. Hamdi, R. Volckmann and P. van Sluis for their support.

Conflict of interest

The authors declare no actual or potential conflicts of interest.

Supplementary material

401_2012_1069_MOESM1_ESM.tiff (20.4 mb)
Supplementary Fig. 1 Hierarchical cluster analysis of mRNA expression of 275 histone modifier genes in primary medulloblastoma (GSE10327) and normal cerebellum (GSE3526) samples revealed dysregulation of many chromatin modifiers in medulloblastoma (TIFF 20839 kb)
401_2012_1069_MOESM2_ESM.tiff (20.4 mb)
Supplementary Fig. 2 Western blot analyses of OTX2 protein levels in time after OTX2 silencing. OTX2 protein levels decrease within 24 hrs to less than 5% of native expression (TIFF 20839 kb)
401_2012_1069_MOESM3_ESM.tiff (20.4 mb)
Supplementary Fig. 3 H3K27 methylation in MYC-bound vs unbound promoters (TIFF 20839 kb)
401_2012_1069_MOESM4_ESM.tiff (20.4 mb)
Supplementary Fig. 4 Examples of differentiation genes and polycomb targets, which show a shift from bivalent to more monovalent marking after OTX2 silencing. Left panels display expression in time of GRIA2, STMN2, SSTR2 and FRMD3 in 3 independent experiments of OTX2 silencing in D425 cells. The right panels display the H3K27me3 and H3K4me3 markings on their promoter region in normal and silenced (72 hrs) cells (TIFF 20839 kb)
401_2012_1069_MOESM5_ESM.xls (92 kb)
Supplementary material 5 (XLS 91 kb)


  1. 1.
    Adamson DC, Shi Q, Wortham M, Northcott PA, Di C, Duncan CG, Li J, McLendon RE, Bigner DD, Taylor MD, Yan H (2010) OTX2 is critical for the maintenance and progression of Shh-independent medulloblastomas. Cancer Res 70(1):181–191. doi: 10.1158/0008-5472.CAN-09-2331 PubMedCrossRefGoogle Scholar
  2. 2.
    Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD (2011) Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA 108(22):E149–E158. doi: 10.1073/pnas.1102223108 PubMedCrossRefGoogle Scholar
  3. 3.
    Bai R, Siu IM, Tyler BM, Staedtke V, Gallia GL, Riggins GJ (2010) Evaluation of retinoic acid therapy for OTX2-positive medulloblastomas. Neuro-oncology 12(7):655–663. doi: 10.1093/neuonc/nop062 PubMedCrossRefGoogle Scholar
  4. 4.
    Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125(2):315–326. doi: 10.1016/j.cell.2006.02.041 PubMedCrossRefGoogle Scholar
  5. 5.
    Bigner SH, Friedman HS, Vogelstein B, Oakes WJ, Bigner DD (1990) Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts. Cancer Res 50(8):2347–2350PubMedGoogle Scholar
  6. 6.
    Boon K, Eberhart CG, Riggins GJ (2005) Genomic amplification of orthodenticle homologue 2 in medulloblastomas. Cancer Res 65(3):703–707 pii:65/3/703PubMedGoogle Scholar
  7. 7.
    Bunt J, de Haas TG, Hasselt NE, Zwijnenburg DA, Koster J, Versteeg R, Kool M (2010) Regulation of cell cycle genes and induction of senescence by overexpression of OTX2 in medulloblastoma cell lines. Mol Cancer Res 8(10):1344–1357. doi: 10.1158/1541-7786.MCR-09-0546 PubMedCrossRefGoogle Scholar
  8. 8.
    Bunt J, Hasselt NE, Zwijnenburg DA, Hamdi M, Koster J, Versteeg R, Kool M (2011) OTX2 directly activates cell cycle genes and inhibits differentiation in medulloblastoma cells. Int J Cancer. doi: 10.1002/ijc.26474 PubMedGoogle Scholar
  9. 9.
    Bunt J, Hasselt NE, Zwijnenburg DA, Koster J, Versteeg R, Kool M (2011) Joint Binding of OTX2 and MYC in promotor regions is associated with high gene expression in medulloblastoma. PLoS ONE 6(10):e26058. doi: 10.1371/journal.pone.0026058 PubMedCrossRefGoogle Scholar
  10. 10.
    de Haas T, Oussoren E, Grajkowska W, Perek-Polnik M, Popovic M, Zadravec-Zaletel L, Perera M, Corte G, Wirths O, van Sluis P, Pietsch T, Troost D, Baas F, Versteeg R, Kool M (2006) OTX1 and OTX2 expression correlates with the clinicopathologic classification of medulloblastomas. J Neuropathol Exp Neurol 65(2):176–186. doi: 10.1097/01.jnen.0000199576.70923.8a PubMedCrossRefGoogle Scholar
  11. 11.
    Di C, Liao S, Adamson DC, Parrett TJ, Broderick DK, Shi Q, Lengauer C, Cummins JM, Velculescu VE, Fults DW, McLendon RE, Bigner DD, Yan H (2005) Identification of OTX2 as a medulloblastoma oncogene whose product can be targeted by all-trans retinoic acid. Cancer Res 65(3):919–924. doi: 65/3/919 PubMedGoogle Scholar
  12. 12.
    Ehrbrecht A, Muller U, Wolter M, Hoischen A, Koch A, Radlwimmer B, Actor B, Mincheva A, Pietsch T, Lichter P, Reifenberger G, Weber RG (2006) Comprehensive genomic analysis of desmoplastic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components. J Pathol 208(4):554–563. doi: 10.1002/path.1925 PubMedCrossRefGoogle Scholar
  13. 13.
    Gibson P, Tong Y, Robinson G, Thompson MC, Currle DS, Eden C, Kranenburg TA, Hogg T, Poppleton H, Martin J, Finkelstein D, Pounds S, Weiss A, Patay Z, Scoggins M, Ogg R, Pei Y, Yang ZJ, Brun S, Lee Y, Zindy F, Lindsey JC, Taketo MM, Boop FA, Sanford RA, Gajjar A, Clifford SC, Roussel MF, McKinnon PJ, Gutmann DH, Ellison DW, Wechsler-Reya R, Gilbertson RJ (2010) Subtypes of medulloblastoma have distinct developmental origins. Nature 468(7327):1095–1099. doi: 10.1038/nature09587 PubMedCrossRefGoogle Scholar
  14. 14.
    Hui AB, Takano H, Lo KW, Kuo WL, Lam CN, Tong CY, Chang Q, Gray JW, Ng HK (2005) Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis. Clinical Cancer Res off J Am Assoc Cancer Res 11(13):4707–4716. doi: 10.1158/1078-0432.CCR-05-0128 CrossRefGoogle Scholar
  15. 15.
    Jones DT, Jager N, Kool M, Zichner T, Hutter B, Sultan M, Cho YJ, Pugh TJ, Hovestadt V, Stutz AM, Rausch T, Warnatz HJ, Ryzhova M, Bender S, Sturm D, Pleier S, Cin H, Pfaff E, Sieber L, Wittmann A, Remke M, Witt H, Hutter S, Tzaridis T, Weischenfeldt J, Raeder B, Avci M, Amstislavskiy V, Zapatka M, Weber UD, Wang Q, Lasitschka B, Bartholomae CC, Schmidt M, von Kalle C, Ast V, Lawerenz C, Eils J, Kabbe R, Benes V, van Sluis P, Koster J, Volckmann R, Shih D, Betts MJ, Russell RB, Coco S, Tonini GP, Schuller U, Hans V, Graf N, Kim YJ, Monoranu C, Roggendorf W, Unterberg A, Herold-Mende C, Milde T, Kulozik AE, von Deimling A, Witt O, Maass E, Rossler J, Ebinger M, Schuhmann MU, Fruhwald MC, Hasselblatt M, Jabado N, Rutkowski S, von Bueren AO, Williamson D, Clifford SC, McCabe MG, Collins VP, Wolf S, Wiemann S, Lehrach H, Brors B, Scheurlen W, Felsberg J, Reifenberger G, Northcott PA, Taylor MD, Meyerson M, Pomeroy SL, Yaspo ML, Korbel JO, Korshunov A, Eils R, Pfister SM, Lichter P (2012) Dissecting the genomic complexity underlying medulloblastoma. Nature 488(7409):100–105. doi: 10.1038/nature11284 PubMedCrossRefGoogle Scholar
  16. 16.
    Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA, Cho YJ, Koster J, Schouten-van Meeteren A, van Vuurden D, Clifford SC, Pietsch T, von Bueren AO, Rutkowski S, McCabe M, Collins VP, Backlund ML, Haberler C, Bourdeaut F, Delattre O, Doz F, Ellison DW, Gilbertson RJ, Pomeroy SL, Taylor MD, Lichter P, Pfister SM (2012) Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol 123(4):473–484. doi: 10.1007/s00401-012-0958-8 PubMedCrossRefGoogle Scholar
  17. 17.
    Kool M, Koster J, Bunt J, Hasselt NE, Lakeman A, van Sluis P, Troost D, Meeteren NS, Caron HN, Cloos J, Mrsic A, Ylstra B, Grajkowska W, Hartmann W, Pietsch T, Ellison D, Clifford SC, Versteeg R (2008) Integrated genomics identifies five medulloblastoma subtypes with distinct genetic profiles, pathway signatures and clinicopathological features. PLoS ONE 3(8):e3088. doi: 10.1371/journal.pone.0003088 PubMedCrossRefGoogle Scholar
  18. 18.
    Lo KC, Rossi MR, Burkhardt T, Pomeroy SL, Cowell JK (2007) Overlay analysis of the oligonucleotide array gene expression profiles and copy number abnormalities as determined by array comparative genomic hybridization in medulloblastomas. Genes Chromosom Cancer 46(1):53–66. doi: 10.1002/gcc.20388 PubMedCrossRefGoogle Scholar
  19. 19.
    McCabe MG, Ichimura K, Liu L, Plant K, Backlund LM, Pearson DM, Collins VP (2006) High-resolution array-based comparative genomic hybridization of medulloblastomas and supratentorial primitive neuroectodermal tumors. J Neuropathol Exp Neurol 65(6):549–561PubMedCrossRefGoogle Scholar
  20. 20.
    McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Iii AD, Diaz E, Lafrance LV, Mellinger M, Duquenne C, Tian X, Kruger RG, McHugh CF, Brandt M, Miller WH, Dhanak D, Verma SK, Tummino PJ, Creasy CL (2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. doi: 10.1038/nature11606 Google Scholar
  21. 21.
    Mendrzyk F, Radlwimmer B, Joos S, Kokocinski F, Benner A, Stange DE, Neben K, Fiegler H, Carter NP, Reifenberger G, Korshunov A, Lichter P (2005) Genomic and protein expression profiling identifies CDK6 as novel independent prognostic marker in medulloblastoma. J Clin Oncol 23(34):8853–8862. doi: 10.1200/JCO.2005.02.8589 PubMedCrossRefGoogle Scholar
  22. 22.
    Northcott PA, Nakahara Y, Wu X, Feuk L, Ellison DW, Croul S, Mack S, Kongkham PN, Peacock J, Dubuc A, Ra YS, Zilberberg K, McLeod J, Scherer SW, Sunil Rao J, Eberhart CG, Grajkowska W, Gillespie Y, Lach B, Grundy R, Pollack IF, Hamilton RL, Van Meter T, Carlotti CG, Boop F, Bigner D, Gilbertson RJ, Rutka JT, Taylor MD (2009) Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma. Nat Genet 41(4):465–472. doi: 10.1038/ng.336 PubMedCrossRefGoogle Scholar
  23. 23.
    Palhan VB, Chen S, Peng GH, Tjernberg A, Gamper AM, Fan Y, Chait BT, La Spada AR, Roeder RG (2005) Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration. Proc Natl Acad Sci USA 102(24):8472–8477. doi: 10.1073/pnas.0503505102 PubMedCrossRefGoogle Scholar
  24. 24.
    Parsons DW, Li M, Zhang X, Jones S, Leary RJ, Lin JC, Boca SM, Carter H, Samayoa J, Bettegowda C, Gallia GL, Jallo GI, Binder ZA, Nikolsky Y, Hartigan J, Smith DR, Gerhard DS, Fults DW, VandenBerg S, Berger MS, Marie SK, Shinjo SM, Clara C, Phillips PC, Minturn JE, Biegel JA, Judkins AR, Resnick AC, Storm PB, Curran T, He Y, Rasheed BA, Friedman HS, Keir ST, McLendon R, Northcott PA, Taylor MD, Burger PC, Riggins GJ, Karchin R, Parmigiani G, Bigner DD, Yan H, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE (2011) The genetic landscape of the childhood cancer medulloblastoma. Science 331(6016):435–439. doi: 10.1126/science.1198056 PubMedCrossRefGoogle Scholar
  25. 25.
    Pugh TJ, Weeraratne SD, Archer TC, Pomeranz Krummel DA, Auclair D, Bochicchio J, Carneiro MO, Carter SL, Cibulskis K, Erlich RL, Greulich H, Lawrence MS, Lennon NJ, McKenna A, Meldrim J, Ramos AH, Ross MG, Russ C, Shefler E, Sivachenko A, Sogoloff B, Stojanov P, Tamayo P, Mesirov JP, Amani V, Teider N, Sengupta S, Francois JP, Northcott PA, Taylor MD, Yu F, Crabtree GR, Kautzman AG, Gabriel SB, Getz G, Jager N, Jones DT, Lichter P, Pfister SM, Roberts TM, Meyerson M, Pomeroy SL, Cho YJ (2012) Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations. Nature 488(7409):106–110. doi: 10.1038/nature11329 PubMedCrossRefGoogle Scholar
  26. 26.
    Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA (2000) Genome-wide location and function of DNA binding proteins. Science 290(5500):2306–2309. doi: 10.1126/science.290.5500.2306 PubMedCrossRefGoogle Scholar
  27. 27.
    Robinson G, Parker M, Kranenburg TA, Lu C, Chen X, Ding L, Phoenix TN, Hedlund E, Wei L, Zhu X, Chalhoub N, Baker SJ, Huether R, Kriwacki R, Curley N, Thiruvenkatam R, Wang J, Wu G, Rusch M, Hong X, Becksfort J, Gupta P, Ma J, Easton J, Vadodaria B, Onar-Thomas A, Lin T, Li S, Pounds S, Paugh S, Zhao D, Kawauchi D, Roussel MF, Finkelstein D, Ellison DW, Lau CC, Bouffet E, Hassall T, Gururangan S, Cohn R, Fulton RS, Fulton LL, Dooling DJ, Ochoa K, Gajjar A, Mardis ER, Wilson RK, Downing JR, Zhang J, Gilbertson RJ (2012) Novel mutations target distinct subgroups of medulloblastoma. Nature. doi: 10.1038/nature11213 Google Scholar
  28. 28.
    Roth RB, Hevezi P, Lee J, Willhite D, Lechner SM, Foster AC, Zlotnik A (2006) Gene expression analyses reveal molecular relationships among 20 regions of the human CNS. Neurogenetics 7(2):67–80. doi: 10.1007/s10048-006-0032-6 PubMedCrossRefGoogle Scholar
  29. 29.
    Rugg-Gunn PJ (2012) Epigenetic features of the mouse trophoblast. Reprod Biomed Online. doi: 10.1016/j.rbmo.2012.01.012 PubMedGoogle Scholar
  30. 30.
    Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34(2):374–378PubMedGoogle Scholar
  31. 31.
    Tan J, Yang X, Zhuang L, Jiang X, Chen W, Lee PL, Karuturi RK, Tan PB, Liu ET, Yu Q (2007) Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev 21(9):1050–1063. doi: 10.1101/gad.1524107 PubMedCrossRefGoogle Scholar
  32. 32.
    Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, Ellison DW, Lichter P, Gilbertson RJ, Pomeroy SL, Kool M, Pfister SM (2012) Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 123(4):465–472. doi: 10.1007/s00401-011-0922-z PubMedCrossRefGoogle Scholar
  33. 33.
    Young MD, Willson TA, Wakefield MJ, Trounson E, Hilton DJ, Blewitt ME, Oshlack A, Majewski IJ (2011) ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity. Nucleic Acids Res 39(17):7415–7427. doi: 10.1093/nar/gkr416 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Jens Bunt
    • 1
  • Nancy A. Hasselt
    • 1
  • Danny A. Zwijnenburg
    • 1
  • Jan Koster
    • 1
  • Rogier Versteeg
    • 1
  • Marcel Kool
    • 1
    • 2
  1. 1.Department of OncogenomicsAcademic Medical CenterAmsterdamThe Netherlands
  2. 2.Division of Pediatric NeurooncologyGerman Cancer Research Center (DKFZ)HeidelbergGermany

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