Somatic Histone H3 Alterations in Pediatric Diffuse Intrinsic Pontine

© 2012 Nature America, Inc. All rights reserved. J.R.D. ([email protected]), Jinghui Zhang ([email protected]) or Jinghui Zhang J.R.D. S.J.B. ([email protected]) ([email protected]), ([email protected]). is provided in the Hospital, Memphis, Tennessee, USA. Institute, Washington University School of Medicine, Saint Louis, Missouri, USA. Hospital, Memphis, Tennessee, USA. 1 a non-BS-PG. is SJHGG043 and DIPGs, are and both G and A and SJHGG001 SJHGG079 for alteration. the p.Gly34Arg alteration p.Lys27Met the T for A and both of presence the N indicates arrows. by marked are and DNA normal matched to compared tumor indicated the in shown are Mutations substitution. a p.Gly34Arg encoding a or substitution a p.Lys27Met encoding mutations representative showing chromatograms sequencing F four subjects had a recurrent somatic adenine-to individuals.Tumorsaffected matched fromseven tissuenormal from performed whole-genome sequencing (WGS) of DNA from DIPGs and than 10% (ref. diagnosis cal of glioblastoma, and has long-terma rate survival of less tumor, arises almost exclusively astrocytic brainstem inaggressive children,an glioma, usuallypontine with intrinsic histopathologi Diffuse H3F3A An caused or contained We and tissue sequencing pontine To Washington University Pediatric Cancer Genome Project Suzanne J Baker James R Downing Richard J Gilbertson Amar Gajjar Li Ding LuCharles Gang Wu glioblastomas gliomas and non-brainstem intrinsic diffuse pontinepediatric Somatic histone H3 alterations in Nature Ge Nature Received 21 December 2011; accepted 9 January 2012; published online 29 January 2012; Department Department of Biology,Computational St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. igure 1 igure

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Hartwell Hartwell Center of and Biotechnology St. Bioinformatics, Jude Children’s Research 78% of DIPGs and 22% of non-BS-PGs ( transversions encoding p.Lys27Met alterationswithDIPG analyzed inbyWGS, wefound recurrent adenine-to-thymine Supplementary Tables 1 43 DIPGs as well as 36 non-BS-PGs (see genes that code for histone H3 isoforms in a we validationperformed targeted cohort sequencing comprisingof the exons encoding Lys27 in all 16 histone H3.1 isoform. related closely the in identified thymine transversion (c.83A>T) encoding a p.Lys27Met alteration was adenine-to- analogous an individual, affected fifth a from tumor the (p.Lys27Met)proteinhistone( H3.3 the in (c.83A>T) in detected inwere genesother H3 histonepediatric 16 the ofbrain any in mutationstumors, no but 3), including(grade 7 low-grade brainstem astrocytomasanaplastic pediatric 9 of 1 p.Lys27Metin alterationthe atric high-grade gliomas. We also detected a 14 genes encoding histone H3 isoforms. mutagenesis. There were no Lys27 or Gly34 mutationsapy, in anyindicating of thatthe otherthis alteration was not secondary to therapy-inducedalteration was found in seven of eight DIPGwere samples somatic obtained in naturebefore ther( germline DNA was found to be wild type, verifying that these mutationsidentified wasmatchedwhichforand normal DNAavailable, was the and6non-BS-PG samples in which an sequencing or SNP arrays in the validation cohort. For 32 DIPG lociusingWGSsamples orSNParrays discoverythein cohort orusing Sanger exclusive. There was no evidence of loss of heterozygosity at thethe mutated50 DIPGs analyzed. These three different in H3 mutations were mutually a new guanine-to-adenine transition resulting in a p.Gly34Arg alteration To determine the frequency of mutations in the histone H3 gene family, The identified alterations in histone H3 seem to be exclusive to pedi H3F3A Normal C SJHGG001 H3F3A C Tumor T T C C d G o G i was identified in 5 of 36 (14%) non-BS-PGs but not in any of C C : (p.Lys27Met) 1 A A A 0 NG H3F3A . G 1 0 A A 3 G G 8 T T 2 / G G n Department Department of Oncology, St. Jude Children’s Research tions n o i at c i n u m m o c f e i r b resulting in a substitution to methionine at lysine 27 C g C . 1 Supplementary Table 1 1 0 and 2 C Normal SJHGG079 HIST1H3B C Tumor T T 2 C C G ). Including the original seven individuals G C C HIST1H3B A A A NG (p.Lys27Met) G A A G G H3F3A C Fig. C 7 G Supplementary Methods G A A full list of consortium members C C Supplementary Fig. 1 Fig.Supplementary gene, which codes for the the for codes which gene, H3F3A 1 and ). Of note, the p.Lys27Met or H3F3A A Normal SJHGG043 H3F3A Tumor A HIST1H3B Table C C mutation encoding T T GG GG or (p.Gly34Arg) A A 1 GGGG 4 NG ). In addition, HIST1H3B The The Genome GG mutation T T

G G A A and ). In ). in - -

© 2012 Nature America, Inc. All rights reserved. monomethylation (H3K27me) is associated with gene activation gene with associated is (H3K27me) monomethylation and cells, especially pluripotent in genes, processes of developmental in involved silencing those the with associated is (H3K27me3) complexes or activator repressor and transcriptional histones modified between interactions direct and state chromatin both affecting by expression the histone tail have an important role in epigenetic regulation of gene tors that bind to histones. Complex post-translational modifications of effec other with or interactions compaction chromatin interactions, histone-DNA affecting by function and structure nucleosome affect may nucleosome, of core the histone globular the exits tail the where site the to close are which residues, invariant these at ity. Alterations accessibil and influ structure chromatin of regulation which dynamic the ences protein, H3 histone the of tail N-terminal conserved highly the within located are residues Gly34 and Lys27 the function, DIPGs of 30% than greater in occur that alterations ( genes or ( of presence the between correlation significant no was there however, and non-BS-PGs DIPGs of in 20% more gain than both mosomal identified in in (see URLs). In fact, no non-silent, coding germline variants were found (NHLBI)exome sequencing project (ESP) that contains 5,400exomes Nationalthe GenomesProject,InstituteinHeart, orLung,Blood and includes1000SNPs39,484,957 submitteddatabase,whichthe 135 by dbSNPthehistone affecting Lys27genesinanythe of H3Gly34in or polymorphismsgermline of occurrences no Furthermore,werethere in the histone H3 loci in any of the tumor subtypes evaluated by WGS. Tables 1 ( tumors pediatric system nervous non–central 170 22 low-grade ependymomas, and 15 mutations were also sequencing, not detected in Sanger an non-brainstemadditional targeted gliomas by evaluated by evaluated WGS, and gliomas 38 medull Gene and G T B a All H3 HIST1H3B H3F3A H3F3A sized and incorporated into nucleosomes during S phase, whereas whereas isoform enriched that is H3.3 is selectively a replication-independent phase, S during nucleosomes into incorporated and sized synthe histones, replication-dependent are H3.2 and H3.1 isoform. p.Lys27Metof the alterations. impact and p.Gly34Arg functional gain-of- a suggesting Additional phenotype. function studies substitutions, are required to the determine acid amino same always the and genes encode H3 histone of family large a of two only in state or However,activation. the mutations are present in the heterozygous repression role of H3 in either the histone may affect which position, loss of as function, it removes the ability to methylate or this acetylate regions active regulated, with acetylated Lys27 being associated with transcriptionally highly also are H3 histone of Lys27 of deacetylation and Acetylation For DIPGs,total P able able 1 = 0.7). There was also no significant association between between association significant no also was There 0.7). = Although it is unclear exactly how these mutations affect histone H3 H3F3A Of Of note, mutations were found more affecting than one histone H3 H3F3A HIST1H3B tions n o i at c i n u m m o c f e i r H3F3A BM P

and = 0.7) or cyclin D family genes, genes, family D cyclin or 0.7) = F , and only one encoded amino acid change (p.Gln20Glu) was is located on chromosome 1q, a region of large-scale chro 1q, a of on region large-scale chromosome is located requency requency of recurrent somatic mutations in D or or 9 . It is notable that trimethylation of Lys27 in histone H3 H3 histone in Lys27 of trimethylation that notable is It . 3 HIST1H3B n ). We also did not find any evidence of structural variations =50. 1 mutation and of amplification kinase receptor tyrosine 1 HIST1H3B . . The replacement of Lys27 implies with a methionine Amino acidchange b For non-BS-PGs,total p.Lys27Met p.Lys27Met p.Gly34Arg in these two public SNP databases. mutations and gain of chromosome 1q 1q chromosome of gain and mutations n =36. DIPG 39 (78) 30 (60) 0 9 (18) CDK4 a ( % ) or or 2 CDK6 Supplementary , non-BS-PG 4 o , 6 blastomas and . 13 (36) I PG PG 5 (14) 7 (19) 1 (3) ( P

= 0.5), 0.5), = H3F3A b ( % 2– 1 ) 8 0 - - - - - ; .

a selective advantage in the unique context of the developing brain brain developing the of context unique the in advantage selective a data from adult development, glioblastomas sequence exome in with identified not associated were mutations H3 genes histone of expression regulating H3 in histone in residue Lys27 the for role a with consistent Perhaps to the report of first our mutations somatic knowledge in histone H3. histones have been identified in a number of different cancers Although mutations in genes encoding post-translational modifiers of tive of regulation genes developmental or telomere or length stability. could potentially affect epigenetic regulation of gene expression, selec non-BS-PG. and DIPG for origin of cells the in part explain why two these genes are targeted for selectively mutation of H3F3A regulation post-translational or transcriptional differential that possible is It H3.1. and H3.3 of regulation in differences the despite HIST1H3B p.Lys27Met in either encoding mutations sive monoallelic However, advantage. and exclu mutually the recurrent selective cific that disruption of the specialized functions of H3.3 may provide a spe telomere stabilization affect can incorporation its where telomeres, at and sites factor–binding transcription and genes transcribed actively within 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturegenetics/. The authors declare no competing interests.financial Zhang and S.J.B. wrote the manuscript. G.W., M.P., Jinghui Zhang and S.J.B. prepared the tables and figures. J.R.D., Jinghui modeling for the mutations. L.D. and Jinghui the Zhangdata analysis. supervised the Sanger sequencing data for the validation cohort. performed structural R.H. T.A.M., B.S.P. and Junyuan Zhang performed validation experiments. J.B. analyzed from other tumor G.W.types. and C.L. analyzed the whole-genome sequence data. histopathological analyses. D.W.E., M.A.D., C.G.M., R.J.G. and J.R.D. provided data A.B. and A.G. provided samples and clinical data. D.W.E. performed S.J.B., T.A.M., B.S.P., C.Q., J.R.D., andE.R.M. R.K.W. the designed experiments. Musicians Against Childhood Cancer and the Noyes Brain Tumor Foundation.the Smile for Sophie Forever Foundation, Tyler’s Treehouse Foundation,Research from the the National Brain Tumor Society, the Cure Starts NowUS Foundation,National Institutes of Health (NIH) (CA096832), the Sydney Schlobohm Chair of Charities (ALSAC) of St. Jude Children’s Research Hospital and by Pediatricgrants fromCancer the Genome Project and the American Lebanese Syrian Associated was supported by the St. Jude Children’s Research Hospital–WashingtonSt. Jude University Children’s Research Hospital and Beckman Coulter Genomics. Thishelpful work discussions, the Hartwell Center of Biotechnology and Bioinformatics at We thank X. Zhu and A. Diaz for assistance with PCR reactions, J. Partridge for Note: Supplementary information is available on the EGAS0000100192. under (EBI), Institute Bioinformatics by European the is hosted which (EGA), Archive Genome-phenome numbers. Accession Archive, phenome ESP,NHLBI URLs. adults. and children in gliomagenesis of biology in underlying the difference significant the and highlights C AUTH A ck O

Given the complex regulatory roles of mutations H3, histone these Given the complex regulatory MPETI Paugh, B.S. Paugh, A. Gajjar, & A. Broniscer, no O dbSNP 135, 135, dbSNP and and wledgme R R C N ON G G FI suggest that a similar gain-of-function effect is possible, possible, is effect gain-of-function similar a that suggest HIST1H3B 1 h et al. et 2 TRIBUTI t . The higher frequency of of frequency higher The . t N p a A : DVANCE ONLINE PUBLICATION ONLINE DVANCE /

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