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. 2009 Apr;41(4):465-72.
doi: 10.1038/ng.336. Epub 2009 Mar 8.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

Paul A Northcott  1 , Yukiko Nakahara, Xiaochong Wu, Lars Feuk, David W Ellison, Sid Croul, Stephen Mack, Paul N Kongkham, John Peacock, Adrian Dubuc, Young-Shin Ra, Karen Zilberberg, Jessica McLeod, Stephen W Scherer, J Sunil Rao, Charles G Eberhart, Wiesia Grajkowska, Yancey Gillespie, Boleslaw Lach, Richard Grundy, Ian F Pollack, Ronald L Hamilton, Timothy Van Meter, Carlos G Carlotti, Frederick Boop, Darrell Bigner, Richard J Gilbertson, James T Rutka, Michael D Taylor
Affiliations

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

Paul A Northcott et al. Nat Genet. 2009 Apr.

Abstract

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

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Figures

Figure 1
Figure 1
The medulloblastoma genome. (a) Global view of regions of gain and loss across the genome in a series of 123 nonoverlapping medulloblastomas genotyped on the Affymetrix 500K SNP array platform. Output from GenePattern SNP Viewer. Regions of gain are red; regions of loss are blue. (b) Summary plot showing the frequency of regions of gain and loss in the medulloblastoma genome. Output from dChipSNP. Recurrent losses are observed on chromosomes 6, 8, 9q, 10q, 11, 16q, 17p and X. Recurrent gains are observed on chromosome 1q, 7 and 17q. (c) GISTIC output shows significant regions of amplification and gain from nonoverlapping cohorts of medulloblastomas analyzed on the 100K (left) and 500K (right) SNP array platforms. Significant regions are labeled by cytoband, and notable genes are identified. (d) GISTIC output shows significant regions of hemizygous and homozygous deletion from nonoverlapping cohorts of medulloblastomas analyzed on the 100K (left) and 500K (right) SNP array platforms. Significant regions are labeled by cytoband, and notable genes are identified. (e) Amplification of MYC family oncogenes in medulloblastoma (MB) in our cohort of 212 medulloblastomas. Output from dChipSNP. Vertical bars denote MYC family gene loci. (f) Rare amplifications of GLI2 and GLI1 in medulloblastoma, downstream effectors of Sonic Hedgehog signaling. Output from dChipSNP. Vertical bars denote the GLI1 and GLI2 loci.
Figure 2
Figure 2
Copy number aberration of genes controlling histone lysine methylation in medulloblastoma. (a) Output from dChipSNP shows focal homozygous deletion limited to EHMT1 in MB-118 and MB-149. MB-101 is diploid for chromosome 9q, whereas MB-160, MB-163, MB-165, MB-166, MB-184, MB-198 and MB-224 are monosomic for 9q. (b) GISTIC output for chromosome 9 (500K SNP array) shows a significant region of focal loss on 9q34 at the EHMT1 locus. (c) Real-time genomic PCR at the EHMT1 locus confirms somatic homozygous deletion in tumor samples, but not in matched constitutional DNA. (d) qRT-PCR for EHMT1 shows significantly decreased expression of EHMT1 in samples with monosomy 9q, as opposed to tumors with diploid chromosome 9q. Two-sample Wilcoxon test, P = 0.0002468. (e) Immunohistochemical staining for EHMT1 expression and H3K9 dimethylation was done on a 64-tumor human medulloblastoma tissue microarray. Staining was graded from 0 to 3 as illustrated. Percentage of tumors in each category is noted below each category. Two-sample test comparing proportions, P = 0.0024. (f) Interphase FISH on paraffin embedded tissues on a medulloblastoma tissue microarray shows amplification of JMJD2C (green) at 9p24.1 as opposed to a control probe (red) at 9q31.2 in a representative medulloblastoma sample. (g) qRT-PCR of JMJD2C shows greater than twofold increased expression in 15% of medulloblastomas as compared to normal fetal cerebellum. Wilcoxon signed rank test, P = 0.1351. (h) qRT-PCR of JMJD2B shows greater than twofold increased expression in 7.5% of medulloblastomas as compared to normal fetal cerebellum. Wilcoxon signed rank test, P = 0.0341. (i) qRT-PCR of SMYD4 shows greater than twofold decreased expression in 30% of medulloblastomas as compared to a normal adult cerebellar control. Wilcoxon signed rank test, P = 2.596e–06. (j) qRT-PCR of BMI1 shows greater than twofold increased expression in >80% of medulloblastomas as compared to normal fetal cerebellum. Wilcoxon signed rank test, P = 9.095e–13. Error bars, ± s.d. See Supplementary Table 6 online for list of primers.
Figure 3
Figure 3
Re-expression of L3MBTL3 in the DAOY medulloblastoma cell line. (a) Output from the UCSC Genome Browser illustrating a homozygous deletion on chromosome 6 that encompasses L3MBTL3, SAMD3 and TMEM200A. Inferred copy number data from SNP array analysis of DAOY was uploaded to the UCSC Genome Browser and is shown in red. (b) GISTIC output for chromosome 6 (100K SNP array) identifies a significant region of extremely focal loss that includes L3MBTL3. (c) Two independent stable transfectants of the DAOY medulloblastoma cell line expressing L3MBTL3 are growth-inhibited as compared to DAOY empty vector, and SAMD3-, TMEM200A- and GFP-expressing controls. (d) Five thousand DAOY cells transfected with either L3MBTL3 or controls were seeded and grown for 7 d. There is greatly reduced growth of the cells re-expressing L3MBTL3 as compared to empty vector control. (e) Overexpression of L3MBTL3 has minimal effect on the growth rate of the D283 medulloblastoma cell line. (f) No sizable difference in the extent of Annexin V labeling is observed in DAOY cells re-expressing L3MBTL3 compared to controls. (g) Flow cytometry analysis of DAOY cells transfected with L3MBTL3 shows a marked reduction in the percentage of cells in G1 as compared to empty vector control. There is also accumulation of cells in S phase of the cell cycle in L3MBTL3 transfectants, as would be predicted in cells with decreased expression from E2F- dependent promoters. (h) Chromatin immunoprecipitation followed by end-point PCR demonstrates that DAOY-L3MBTL3 transfectants show increased H3K9 dimethylation in the promoter regions of the E2F6 target genes MYC, CDC25A and TK1 as compared to controls. Error bars, ± s.d.
Figure 4
Figure 4
H3K9 in the developing external granule cell layer. (a) Hematoxylin and eosin (H&E) staining of the P7 murine cerebellum. The external layer of the EGL (ext-EGL), the internal layer of the EGL (int-EGL), the molecular layer (ML), and the subarachnoid space (SAS). Original magnification ×400; scale bar, 50 microns (mu). (b) EHMT1 staining of an adjacent section of the external granule cell layer of the cerebellum. EGL cells are a putative cell of origin in medulloblastoma. (c) Dimethylation of histone 3, lysine 9 (H3K9me2) is seen to be more extensive in the inner, postmitotic layer of the cerebellum, with very little staining in the outer, highly proliferative layer of the EGL. (d) Expression of the cell cycle arrest protein p27Kip1 colocalizes with H3K9me2 in the inner EGL. (e) Monomethylation of H3K9 is not seen by immunohistochemistry in the P7 cerebellum. (f) Rare immunohistochemical staining for H3K9me3 is found in a small subset of mitotic cells of the P7 EGL. (g) Retroviral infection of P7 EGL cells with WZL-GFP shows high efficiency of transduction (infection rate >50%), but only rare cells infected with WZL-HA-JMJD2C could be found (infection rate <1%). EGL cells expressing HA-JMJD2C have decreased levels of H3K9 dimethylation. (h) Viral infection of NIH3T3 cells shows high levels of transduction for both WZL-GFP and WZL-HA-JMJD2C. (i) Viral infection of the medulloblastoma cell line UW228 shows high levels of transduction for both WZL-GFP and WZL-JMJD2C. α-GFP, antibody to GFP DAPI, 4,6-diamidino-2-phenylindole.
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