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

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Abstract

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.

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Acknowledgments

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.

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  1. Marcel Kool

    Present address: Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany

Authors and Affiliations

  1. Department of Oncogenomics, Academic Medical Center, Amsterdam, The Netherlands

    Jens Bunt, Nancy A. Hasselt, Danny A. Zwijnenburg, Jan Koster, Rogier Versteeg & Marcel Kool

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Correspondence to Marcel Kool.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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

Supplementary Fig. 2 Western blot analyses of OTX2 protein levels in time after OTX2 silencing. OTX2 protein levels decrease within 24 h to less than 5 % of native expression.

Supplementary Fig. 3 H3K27 methylation in MYC-bound vs unbound promoters.

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 h) cells.

401_2012_1069_MOESM1_ESM.tiff

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

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)

Supplementary Fig. 3 H3K27 methylation in MYC-bound vs unbound promoters (TIFF 20839 kb)

401_2012_1069_MOESM4_ESM.tiff

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)

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Bunt, J., Hasselt, N.A., Zwijnenburg, D.A. et al. OTX2 sustains a bivalent-like state of OTX2-bound promoters in medulloblastoma by maintaining their H3K27me3 levels. Acta Neuropathol 125, 385–394 (2013). https://doi.org/10.1007/s00401-012-1069-2

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