© 2016 Nature America, Inc. All rights reserved. A full list of affiliations appears at the end of the paper. to specific was rearrangement this that and member family MYB a MYB newly generated and published PLGG genomic data sets they defined. be to which yet are by gliomagenesis to contribute mechanisms the and system nervous central the in factors transcription family MYB of oncogenicity the Furthermore, of a bor of ously identified one angiocentric glioma with deletion of the 3 and ependymomas features with astrocytomas tological unknown. are subtypes histological with associations PLGGs in events rare as in and in MYB genes family Health pathway (MAPK) protein kinase in alterations the mitogen-activated World frequent undergo PLGGs tumor. of brain pediatric common most the group heterogeneous Organization (WHO) a grade I and II tumors that collectively represent encompass PLGGs of aberrant rearrangements specific analysis Angiocentric Resnick C & Adam Angela J Waanders Bradner E James Sabine Mueller Almos Klekner Mirko Scagnet Sara Seepo Pascale Varlet E Henn Rosemary Shakti H Ramkissoon Rhamy Zeid Bandopadhayay Pratiti mechanism a tripartite through tumorigenicity MYB NATURE NATURE GE Received 21 August 2015; accepted 6 January 2016; published online 1 February 2016;

MYB MYB To address these questions, we performed a combined analysis of analysis combined a performed we questions, these Toaddress his share and cortex cerebral the in arise gliomas Angiocentric MYB a

single - MYB QKI 1 are heterogeneous; several fusion partners have been reported alterations in angiocentric glioma has not been determined. determined. been not has glioma angiocentric in alterations

, , and one other angiocentric glioma has been reported to har and of

MYB-QKI rearrangement to be the most common event involving involving event common most the be to rearrangement

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154 tumors (90%), including all 140 tumors subjected to whole-genome Note Supplementary seq data ( genetic somatic eventssamplesacrosswhole-genomeallwith sequencingRNA-or recurrent significantly of analyses performed We samples published ( subtypes histological ten spanning PLGGs 172 from data (RNA-seq) RNA sequencing and/or performed a combined genomic analysis of whole-genome sequencing we this, To address aberrations. recurrent detect to power statistical achieve to subtypes histological rare of numbers sufficient contain Previously published genomic analyses of PLGGs did not individually Angiocentric RESULTS gene. suppressor MYB translocation driving selectively loop enhancer feedback autoregulatory an protein, establishes that fusion generation MYB-QKI mechanisms: oncogenic three of through oncogenicity to tributes con rearrangement this that found Wealso gliomas. angiocentric MYB 2

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© 2016 Nature America, Inc. All rights reserved. rearrangement, or 3 tric glioma. None of the 147 non-angiocentric gliomas profiled with with profiled gliomas non-angiocentric 147 None the of glioma. tric sufficient with scoring. quantitative for tumors cells of numbers five of two in analysis FISH with was observed cells) tumor of ~50% in (aberration heterogeneity of evidence event genetic appeared to be present in the majority of although cells, to of partner fusion the which array or FISH displayed sequencing, CGH whole-exome RNA-seq, sequencing, 2 Fig. ( CGH) (arrayhybridization comparativegenomic array and/or sequencing by whole-exome were analyzed gliomas tric detect by to FISH were analyzed gliomas Nine angiocentric assays. targeted using available tissue paraffin-embedded formalin-fixed, only with glioma. For we validation, studied 12 gliomas angiocentric additional partner, and we were by struck two new findings: not was which glioma, a contained reviewed, centrally angiocentric other The rearrangements. in resulting deletions intrachromosomal review, exhibited pathology central to subjected tumors all including gliomas, centric ( gliomas and angiocentric astrocytomas diffuse predominantly tumors (10%), MYBL1 (83%; tumors 129 in observed were alterations or structural Rearrangements sequencing. profiles from whole-genome sequencing data for and whole-genome sequencing alone ( angiocentric glioma. The The dashed box highlights angiocentric gliomas.( NA. designated are unavailable is histology Tumorswhich for (NOS). specified otherwise not PLGG and (PXA) xanthoastrocytoma pleomorphic (GG), ganglioglioma (DNT), tumor neuroepithelial dysembryoplastic (DA), astrocytoma diffuse (OD), oligodendroglioma (AG), glioma angiocentric (PMA), astrocytoma (PA), pilomyxoid astrocytoma pilocytic included subtypes Histological report). this to unique tumors and sequences RNA-seq (analysis includes both published and/or sequencing whole-genome with profiled PLGGs 172 of 154 in identified were alterations MYB Figure 1 ARTICLES 2 exhibited RNA-seq or sequencing whole-genome a NTRK2 MYB MYB whole-genome by profiled gliomas angiocentric 19 all total, In Although involving Rearrangements QKI QKI and and Sequencing ). All 12 harbored harbored 12 All ).

or MYB . In tumors confirmed to harbor or MYB-QKI Hist in angiocentric gliomas. Sequence across the breakpoints, as determined by RNA-seq, is shown for each rearrangement. ( rearrangement. each for shown is RNA-seq, by determined as breakpoints, the across Sequence gliomas. angiocentric in

- Location ) were the second most recurrent alteration, affecting 16 16 affecting alteration, recurrent most second the were ) MYBL1 NTRK3 Hist FGFR1 QKI Recurrent rearrangement involving involving rearrangement Recurrent Sour QKI Fig. 1 Fig. BRAF ology TP53 IDH1 NF1 QK one rearrangement or deletion ( deletion or rearrangement ce in angiocentric gliomas. ( gliomas. angiocentric in MYB MYB rearrangements appeared to be specific to angiocen I a and and MYB rearrangements have been described in PLGGs in described been have rearrangements - `

QKI MYB Fig. 1 Fig. Supplementary Fig. 1 Fig. Supplementary alterations, and, in six of the seven cases in in cases seven the of six in and, alterations, fusions were nearly universal in angiocentric in angiocentric universal were nearly fusions P deletion. Scale bars, 5 5 bars, Scale deletion. MYB value represents enrichment of of enrichment represents value a and and MYB aberrations. n MYB MYB = 1), whole-genome sequencing and RNA-seq ( RNA-seq and sequencing whole-genome 1), = Supplementary Table 1 Supplementary could be detected, detected, be could QKI a family members ( -

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alterations and and alterations MYB harbored alterations of either filed with whole-genome sequencing and/or RNA-seq, 10% of tumors Across the entire astrocytomas. cohort of were diffuse 172 tumors pro involving rearrangements and astrocytomas pilocytic supratentorial three in involving rearrangements observed also we ( seq, RNA-seq by transcripts sequencing data ( mRNA whole-genome in genes these in breakpoints fusion number copy observed identified We in result to predicted protein,in-framefusionexpressionwere anMYB-QKIthe( of rearrangements the all 15; intron to 9 QKI of partners fusion the characterize to unable detect to able although sequencing, exome whole- and CGH arrayassays, FISH astrocytomas.The diffuse all ing or array CGH exhibited alterations of sequenc by whole-exome evaluated tumors Five institution. referring but research glioma atreview as had angiocentric the diagnosed been 1 Table tric gliomas; in alterations tumors exhibited these of one Only CGH. array and/or sequencing whole-exome by evaluated gliomas non-angiocentric 55 and FISH by analyzed mas glio non-angiocentric ten cohorts: separate two from PLGGs 65 ( MYB proteins are transcription factors characterized by highly highly lular proto-oncoprotein counterpart c-MYB by comprises an N terminus characterized factors as DNA-binding v-Myb First identified motifs. conserved transcription are proteins MYB 1 P 5 < 0.0001; 0.0001; < In the cohort analyzed by whole-genome sequencing In and/or RNA-by the sequencing cohort analyzed whole-genome All QKI , whereas the n QKI QKI

= 2), or RNA-seq alone ( alone RNA-seq or 2), = and MYB ex ). This tumor was designated not otherwise specified upon upon specified otherwise not designated was tumor This ). ex ex ons 5–8 MYB ons 5–8 MYB ons 5–8

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MYBL1 QKI ). We also evaluated Sour brain ial This r Zhang Jones NA PXA GG DNT DA e Chr (Mb) ce rearrangements in diffuse astrocytoma and and astrocytoma diffuse in rearrangements . epor breakpoint varied in its position from intron intron from position its in varied breakpoint 6 et al e log (copy number ratio) et al

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© 2016 Nature America, Inc. All rights reserved. peak regions of significant deletion in low-grade glioma and bladder and bladder glioma in low-grade deletion of significant regions peak larger in also was It carcinoma. cell squamous cervical and cinoma ( glioblastoma adult in peak a in deletion from The Cancer Genome Atlas (TCGA) cer glioblastoma including cancers, human of number of Deletions system. differentiation godendroglial oli in role essential an has which Quaking, RNA-bindingprotein MYB expression in mouse progenitor cells but not in cortical brain ( zone subventricular region subventricular ( eminence ganglionic the of cells progenitor ( gestation of weeks 22 at eminence ganglionic the from generated cells progenitor neural ( of adult human frontal cortex and white matter was negative for MYB or esophagus blood, skin ( cortex and substantially lower than sues tis normal for WeRNA-seqoccur.data examinedgliomas centric protein. MYB the of tion introns 9 and 15 of terminus are oncogenic transforming weakly in negative domain C-terminal regulatory a and breakpoints domain activation transcriptional different a by to followed motifs DNA-binding (HTH) corresponding helix-turn-helix contains that depicted are MYB-QKI of variants Two adult human glioblastomas. ( ( ependymal/subventricular zone layer of the adult mouse brain. Scale bar, 100 periventricular region of adult mouse brain. Scale bar, 100 subventricular zone but not the ventricular zone of E14.5 mouse ganglionic eminence. Scale bar, 5 Scale bar, 50 ganglionic eminence. The asterisk indicates the region presented at higher magnification in (GE-SVZ) zones. Scale bar, 50 subventricular and (GE-VZ) ventricular including (GE), eminence ganglionic mouse E14.5 mouse brain. Scale bar, 500 from 100 bar, Scale gestation. of weeks 22 at eminence ganglionic the from generated cells stem matter. Scale bar, 100 frontal cortex. Scale bar, 100 per kilobase of transcript per million. ( ( expression (mean) in normal human colon ( Figure 2 NATURE NATURE GE n y Fig. Fig. 2f Fig. Fig. 2b a axis) in adult human glioblastomas. G score refers to GISTIC score GISTIC to refers score G glioblastomas. human adult in axis) = 51), esophagus ( esophagus 51), =

QKI neural 14.5 (E) day embryonic in expressed was MYB mice, In angio where cortex, brain postnatal the in expressed not is MYB MYB
> 1    8 1 M  d and gastric cancer gastric and 4    m. ( . Scale bar, 100 and found that that found and encodes the STAR (signal transduction and activation of RNA) – (14 4&-0

, i c ). In adult mice, we detected expression of MYB in the ependyma/ Alterations of 6* e ); ); however, we detected high MYB expression in human fetal : ) MYB immunohistochemistry demonstrates positive staining in a subset of cells cells of a subset in staining positive demonstrates immunohistochemistry ) MYB N 

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MYB 9 n . Full-length MYB is non-transforming or only only or non-transforming is MYB Full-length . ADVANCE ONLINE PUBLICATION = 38), skin ( skin 38), = M QKI in in vitro .11) M MYB i. 2j Fig. MYB m. ( m. ( Fig. 2d Fig. 10–  1 512,&+75 are predicted to result in C-terminal trunca C-terminal in result to predicted are 9 . . In copy of number analyses cancers 10,570 have been suggested to be oncogenic in a in oncogenic be to suggested been have 1 and and Fig. Fig. 2 d f 3 m expression was negligible in human was expression negligible brain ) Hematoxylin and eosin staining of a sagital section from from section a sagital of staining eosin and ) Hematoxylin . ) Hematoxylin and eosin staining of human fetal neural neural fetal human of staining eosin and ) Hematoxylin M M MYB , ) Structures of the MYB-QKI fusion proteins. The C terminus of QKI includes QUA2 domains. MYB-QKI5 retains an NLS. NLS. an retains MYB-QKI5 domains. QUA2 includes QKI of C terminus The proteins. fusion MYB-QKI the of ) Structures 1 1 m. ( k 0 m. ( 6 , , osset ih rvos eot of reports previous with consistent ), , but MYB proteins with a truncated C , but a with MYB proteins truncated and is widely expressed in the nervous QKI e ?-0 ). a c ). Likewise, immunohistochemistry immunohistochemistry ). Likewise, n M h - ) MYB immunohistochemistry on human adult white white adult human on immunohistochemistry ) MYB occur frequently in human cancers. ( cancers. human in frequently occur = 25) and brain cortex ( cortex brain and 25) = m. ( QKI ) MYB immunohistochemistry on the E14.5 mouse mouse E14.5 the on immunohistochemistry ) MYB b MYB ) MYB immunohistochemistry on human adult adult human on immunohistochemistry ) MYB h f bc 4 g breakpoints mapping between 106&.
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© 2016 Nature America, Inc. All rights reserved. nrae epeso ws o seii t tmr with tumors to specific not was expression increased ( brain normal to compared as activation way path MYB with associated genes of expression increased exhibited without PLGGs ( brain pediatric normal than signature MYB-QKI the activity. of level Fig. 3b Supplementary MYBtr for construct the with in mim-1 induction greatest The vector. control the to compared as MYB in We MYB cells. into HEK293T a induction slight full-length observed together with construct encoding human MYBtr mim-1 from sites chicken the MYB binding known using construct reporter We motifs. of binding binding MYB consensus a generated luciferase ( enhancers overlapped sites genes at downregulated binding MYB-QKI the of 70% only whereas peak, enhancer marked associated with an upregulated gene was associated with an H3K27ac- the signature ( in genes downregulated the of (10/25) 40% only but signature QKI upregulatedMYB- the theof (22/25) ingenes 88% of kb 100within to endogenous the enhancers H3K27ac-defined tic leukemia (T-ALL), where MYB binding was highly correlated with areThese findings consistent with reports in T cell acute lymphoblas ( sites, binding MYB (52% sites 1,907 of lap sites 3,672 was and present at H3K27ac sites, with an 9,122 over motif) binding bound MYB-QKI5 10 regions. × 1 enhancer that of location found the We defines lysine which at H3 (H3K27ac), histone of 27 acetylation against antibody another and MYB of terminus N the recognizes that antibody one using fusion protein, MYB-QKI expressing mNSCs in (ChIP-seq) sequencing lel Cdk6 expression of the fusion protein ( the with most correlated expression differential whose genes 50 the of the RPKM values for each gene in the signature. ** Expression of the signature within each tumor was calculated as the sum Bars represent mean expression of the signature in tumors ( pediatric brain samples ( * shown represent the means of three independent measurements values The cells. HEK293T in MYB full-length or MYBQKI6 MYB-QKI5, (MYBtr), 1–9 exons by encoded MYB truncated express to transfection genes. ** ChIP-seq binding for upregulated ( ( bin. per reads of units in are intensities Scaled signal. MYB-QKI by rank-ordered are regions The peak. a MYB-QKI on centered is row Each ( expression. low indicates blue and expression, high indicates Red control. eGFP expressing cells to relative mNSCs in signature expression MYB-QKI its molecular effects are observed in angiocentric gliomas. ( Figure 3 ARTICLES 4 n c b P ) Percentage of MYB-QKI signature genes with evidence of MYB-QKI MYB-QKI of evidence with genes signature MYB-QKI of ) Percentage = 8) and angiocentric gliomas with with gliomas angiocentric and 8) = ) Heat maps of H3K27ac and MYB-QKI signal in MYB-QKI regions. regions. MYB-QKI in signal MYB-QKI and H3K27ac of maps ) Heat < 0.05, *** 0.05, < Angiocentric gliomas exhibited significantly higher expression of expression higher significantly exhibited gliomas Angiocentric through transcription activate can protein fusion MYB-QKI The MYB-QKI5 binding sites ( paral with immunoprecipitation chromatin performed We comprising signature expression gene MYB-QKI a defined We mim-1 , previously reported to be associated with MYB activation MYB with to associated be , reported previously promoter activity was observed upon cotransfection of cells cells of cotransfection upon observed was activity promoter −7 target promoter

P ) across the genome (92% of these sites contained a MYB MYB a contained sites these of (92% genome the across ) MYB-QKI fusion protein functions as a transcription factor, and and factor, a transcription as functions protein fusion MYB-QKI promoter activity with transfection to express full-length full-length express to transfection with activity promoter < 0.001, paired paired 0.001, < P P < 0.001. ( 0.001. < < 0.001; 0.001; < MYB Myb - n QKI = 8), PLGGs without without PLGGs 8), = promoter ( promoter ), with MYBtr ), with 8 Fig. Fig. 3 e and cotransfected this reporter construct construct reporter this cotransfected and ) Expression of the MYB-QKI signature in normal t test. ( test. alterations ( alterations P 2 -value threshold = 1 × 10 3 c . . We MYB-QKI binding also identified ). Each ). of Each the binding MYB-QKI sites exons1–9 Fig. Fig. 3 n d = 25) and downregulated ( downregulated and 25) = MYB ) mim-1 Supplementary Fig. 3a Fig. Supplementary a exons1–9 - ). These genes included QKI or MYB-QKI ( P = 0.0011) ( 0.0011) = reporter induction following following induction reporter rearrangement ( rearrangement MYB resulting in the highest in highest the resulting P - < 0.0001) ( QKI P exons1–9 P P = 0.0003), but this this but 0.0003), = < 0.01. < -value threshold = threshold -value rearrangement o P Fig. 3 Fig. s.e.m. −5 = 0.003). = P , MYB-QKI or a ) were located = 0.001) and = 0.001) ) Human ) Human i. 3 Fig. n = 4). MYB n e ). ). PLGGs PLGGs ). = 25) = 25) o Fig. 3 Fig. s.e.m. s.e.m. Kit d

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breakpointsofexonsand10 between 9 allele.Three angiocentric gliomas harbored cated and corresponds to the exons retained in the rearranged The (FGFR) gene alterations ( ( higher cantly with gliomas Angiocentric of MYB for the signatureMYB-QKI genes ( rearrangement and was of lower magnitude than the difference observed P a = 0.0062) or PLGGs with with PLGGs or 0.0062) =

truncated MYB - eGFP QKI transcript that is expressed in angiocentric gliomas is trun

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drive Lrrc8c Slc9a3r1 Ankrd28 Frmd4b Sncaip Prune2 Srgap1 Piwil4 Dgkg Tmod1 Uaca Stom Mylip Kif26b Itm2a Lrrc8b Flnb Anks1a Cdk6 Sh3rf3 Zmynd10 Prelp Kit Fras1 Anks1a Lrrc61 Ptpre Tnfrsf21 Bche Gjc3 Lrig1 Smoc1 Mtss1l Igsf21 Plxna2 Atp1a2 Ankh B3gat1 Hepacam Acap3 Nxph1 Ednrb Rev3l Xylt1 Cspg4 Kcnma1 Lifr Sox8 Ptgds Ttyh1 - QKI or fibroblast growth factor receptor Fig. 4 Fig.

Supplementary Fig. 3c Fig. Supplementary aberrant rearrangement exhibit signifi exhibit rearrangement MYB b d c e

a Expression (RPKM sum) Normalized luciferase units DNA peaks ranked by 1,000 1,500 and and 0 binding of MYB-QKI 0.05 0.10 0.15 0.20 500 Reads/bin MYB . These tumorsThese .exhibited MYB 0 0

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© 2016 Nature America, Inc. All rights reserved. otcl ri samples brain cortical tion transloca enhancer from result can expression oncogene Aberrant MYB-QKI aberrant regulation of the expression of truncated remaining the of harbor increased expression of represent means or rearrangement ( expression levels (in RPKM) of tumors with which MYB-QKI binds to the in circuit feedback autoregulatory an and translocation of Figure 4 NATURE NATURE GE associated enhancer present in the ENCODE data from normal brain. of H3K27ac-associated enhancer elements in are associated with H3K27ac (per kb) in and mNSCs. Bottom track (red), MYB-QKI binding within the to PLGGs that harbor a position (Mb) Chr b position (Mb) Chr a H3K27ac RNA H3K27ac H3K27ac BRAF MYB MYB MYB expression (RPKM) 10 20 30 40 . . QKI 2 0 A A 6 6 4 -seq G2 enhancerpeaks G1 enhancerpeaks QKI . In published H3K27ac enhancer profiles from normal human from normal profiles enhancer . In H3K27ac published Normal . The enhancer maps shown are derived from ENCODE data for normal human brain (frontal and temporal lobes). Q3E1 represents a H3K27ac- - MYB QKI loci in human frontal and temporal lobes (Encyclopedia of DNA Elements, ENCODE). Values shown depict the mean number of nucleotides that that nucleotides of number mean the depict shown Values ENCODE). Elements, DNA of (Encyclopedia lobes temporal and frontal human in loci - or FGFR-driven PLGGs (

Angiocentric gliomas exhibit aberrant expression expression aberrant exhibit gliomas Angiocentric SE peaks SE peaks A A G2 H3K27ac G1 H3K27ac BRA

MYB-QKI due to H3K27ac-associated enhancer enhancer H3K27ac-associated to due HBS1L N rearrangement 1 - 35.3 MY 16 ETICS QKI F Non- 4 n M5E1 -duplicat ** B o = 5) relative to normal brain ( brain normal to relative 5) = - AG AG QKI s.e.m. ** s.e.m. rearrangement ( rearrangement MYB tumor

** 1 2 1 MY 64.3 BRAF (0.45) (0.25) ADVANCE ONLINE PUBLICATION ed tumor B exons ( - MYB Q3SE1 MY QKI 2 5 alterations ( alterations , the the , MY P B 1 MYB 35.5

< 0.01 .( 0.01 < M5E1

ex b B results exons 1–9 relative to PLGGs that did not

ons 1–9 Expression relative to Fig. 4 Fig. n promoter. ( promoter.

MYB PLGGs without MYB-QKI = 10). Values shown shown Values = 10). 800 200 400 600 P < 0.05) but had minimal expression 0

in b b n 1 locus is not associated with with associated not is locus ) Exon-specific expression of ). These data support selective, support data These ). MYB = 4). Values shown represent means means represent shown Values 4). = AHI1

enhancer 2345 MYB MYB Q3SE2 a and and ) n MYB 1 - - = 10) = 10) 64.55 QKI QKI LINC00271 1 6 MY QKI

35.8 78

MYB

, with translocation of genomic enhancers from the 3

B

translocation

across both locations locations both across exon c

Breakpoint 9

Luciferase arbitary units Qk 12 10 via MYB-QKI. via 40 80

0 0 locus in mNSCs. ChIP-seq binding peaks are shown. ( shown. are peaks binding ChIP-seq mNSCs. in locus 11 QKI pr 1 12 Co d 63.8 omot

13

ex H3K27ac signal/kb ntr U87 with U87 100 200 300 400 14 QKI ons 5–8 ol 0 er 15 MYB MY and Q3E1 c pr Co MY 16 ** omot B - Q3E1 **

4 in angiocentric gliomas that harbor harbor that gliomas angiocentric in ntr B * - ol o QKI er active active enhancer elements that are translocated proximally toward the expressing gliomas angiocentric the of kb 15 only within the from elements enhancer H3K27ac-associated QKI 3 the at sequencesincluding peaks, H3K27ac several with contrast, In expressed. not that finding the with consistent peaks, enhancer H3K27ac s.e.m. ( s.e.m. QKI o s.e.m. ( s.e.m. Profiling of H3K27ac-associated enhancers for two human human two for enhancers H3K27ac-associated of Profiling pr ( omot MY Fig. Fig. 4c ** e 9 kb B n er ** = 1 ChIP-seq map for each location). * location). each for map ChIP-seq 1 = MY c ** ) Top track (green), H3K27ac binding within the ** Q3E1 1 B and Q3E1 Q3SE1 pr – 64.4 e omot MY Q3SE2 ** ). 15 kb B MYB er - QKI with o from the MYB and without the Q3E1 enhancer cloned into the cells and U87 cells overexpressing MYB-QKI with transfection of the enhancer. ( angiocentric gliomas (AG1 and AG2). SE, super- of ( glioma harboring by RNA-seq, is depicted for the angiocentric MYB MYB pilocytic astrocytoma. The breakpoint for the (M5E1), which is not detected in the aberrant enhancer formation at the of (Q3SE1 and Q3SE2) are located within 500 kb the 3 (bottom). Q3E1 is an enhancer associated with glioma harboring pilocytic astrocytoma (top) and an angiocentric proximity to MYB with an aberrant enhancer associated with the the translocation of H3K27ac-marked enhancers Figure 5 MYB 255 kb b MYB s.e.m. of three individual replicate experiments ) Super-enhancers associated with the 3 ` QKI QKI region of of region rearrangement is to rearrangement predicted bring these QKI and exons 5–8 of of 5–8 exons and - - ( promoter. n ` QKI luc UTR of of UTR promoter ( promoter = 5 each. ** each. 5 = (Q3SE1 and Q3SE2), presented for two . Angiocentric gliomas are associated with promoter ( promoter MYB

construct. Changes in luciferase activity MYB , which is expressed, was associated associated was expressed, is which , rearrangement lies between exons 1–9 of MYB Human angiocentric gliomas exhibit d c - ) H3K27ac signal within the ) MYB QKI QKI QKI MYB - QKI luc - QKI a rearrangement ( rearrangement and and to within 15 kb of the 5 the of kb 15 within to MYB MYB ) H3K27ac enhancer peaks in reporter are shown as the means . Two super-enhancer clusters promoter activation following MYB Fig. Fig. 5 P Fig. 4 Fig. < 0.01. < confirmed the presence of QKI QKI - - QKI QKI - luc P . Expression, as determined a in a a in < 0.05. ( 0.05. < e rearrangement. rearrangement and and Q3E1 construct into U87 ). ARTICLES BRAF 3 Qk Supplementary Supplementary ` end of H3K27ac frontal H3K27ac temporal n MYB = 3) relative relative = 3) Qk Chr. 17q e -duplicated BRAF ) Predicted ) Predicted locus in locus promoter ` H3K27ac MYB-QKI ` MYB end of of end MYB end QKI ` -driven

end end

to is is 5

© 2016 Nature America, Inc. All rights reserved. suppression signature in normal human brain ( shLacZ, control shRNA targeting experiments independent four of means the represent suppression of wild-type mouse with control eGFP or MYB-QKI6 MYB-QKI5, MYB, truncated human expressing mNSCs of and thus are not shown. ( QKI means BRAF angiocentric gliomas ( ( expression of Figure 7 wild-type remaining the of expression the regulate of expression regulating full-length of is enhancer M5E1 truncated aberrant the suggesting that rearrangement, the in retained those to corresponding of exons nine first the of expression showed RNA-seq (M5E1). the with associated peak H3K27ac large a observed ( at peaks formation enhancer the H3K27ac of of observe not evidence did we show indeed, and, not enhancers, does related brain cortical human Normal in promoter the toward proximally translocated are enhancers these rearrangement, QKI 3 region kb 10 of the within enhancers We observed also astrocytoma. a in and brain human mal 3 the with ated Fig. 4 expressing eGFP, which mNSCs never with develop tumors (* injected mice to comparison in latency short with tumors develop that MYB-QKI6 or MYB-QKI5 MYB, truncated overexpressing mNSCs MYB-QKI5 or MYB-QKI6. Scale bars, 2 mm (top) and MYB, 50 eGFP, truncated expressing mNSCs with injections striatal after brain mouse (SCID) immunodeficient combined severe of analysis eosin and of five measurements is depicted. Error bars, s.e.m. Representative images are shown of intracranial mNSC–truncated MYB tumors. ( ( s.e.m. bars, Error depicted. are pools independent five of means The control. eGFP or (short) MYB-QKI6 (short), MYB-QKI5 overexpress that mNSCs of for intracranial mNSC-MYB-QKI6 tumors. ( shown are images Representative s.e.m. bars, Error depicted. are MYBtr MYB, overexpressing cells NIH3T3 of injections flank are depicted. Error bars, s.e.m. ( MYBtr truncated or eGFP overexpressing mNSCs of baseline) to oncogenic. ( Figure 6 ARTICLES 6 was calculated as the sum of the RPKM values for each gene in the signature. d a Supplementary Fig. 5 Fig. Supplementary a `

) Exon-specific expression of to ) Tumor growth following flank injections of NIH3T3 cells overexpressing MYB, MYB-QKI5 (long), MYB-QKI6 (long) or vector control. The mean mean The control. vector or (long) MYB-QKI6 (long), MYB-QKI5 MYB, overexpressing cells NIH3T3 of injections flank following ) Tumor growth

We observed an aberrant enhancer associated with the the with associated enhancer aberrant an observed We Proliferation 0 2 4 6 showed a high number of duplicate reads reads duplicate of number a high showed MYB (Q3SE1 and Q3SE2). In angiocentric gliomas with -driven PLGGs ( QKI exons1–15 exons1–9 ). ChIP-seq analysis identified multiple H3K27ac peaks associ P o <0.000

s.e.m. RNA-seq data for exon 8 of exon for data RNA-seq s.e.m. MYBtr eGFP

MYB-QKI fusion protein and truncated MYB are are MYB truncated and protein fusion MYB-QKI MYB 0 promoter ( promoter and a larger cluster of super-enhancers 100–500 kb 3 Days MYB a o ) . The mean values for five independent pools pools independent five for values mean . The QKI ex s.e.m. * s.e.m. - 1 or a vector control. The means of five measurements measurements five of means The control. a vector or af In vitro QKI ons1–9 MYB MYB t ` er injection from the rearranged allele. The lack of expression expression of lack The allele. rearranged the from , a tumor-suppressor gene. gene. , a tumor-suppressor 3 end of rearrangement disrupts disrupts rearrangement n - indicates that the aberrant enhancer does not not does enhancer aberrant the that indicates cell proliferation (number of cells relative relative cells of (number proliferation cell n Fig. 5 Fig. QKI P = 5). Values represent represent Values = 5). = 5) relative to relative = 5) < 0.05, ** 0.05, < b ). However, in both angiocentric gliomas, we gliomas, angiocentric However, both ). in 5 QKI ) Cell proliferation proliferation ) Cell dfnd nicnrc loa ( glioma angiocentric –defined b BRAF ). b

, similar to the peaks observed in nor in observed peaks the to similar , 3 QKI

Qk Tumor volume (cm ) b

lacZ 0 1 2 3 4 ) Tumor growth following following ) Tumor growth . Values . Values in P -duplicated supratentorial pilocytic pilocytic supratentorial -duplicated MYB 11 P < 0.01. < 0.01. =0.00 ; shQKI, shRNA targeting Days R MY MYBtr

etr 21 B in the pilocytic astrocytoma astrocytoma pilocytic the in o af 13 viral contr c

92 ex t ) er injection

ons1–1

In vitro

n 63 = 8) and angiocentric gliomas ( gliomas angiocentric and 8) = P 5 < 0.05). < ol 34 cell proliferation proliferation cell a

Expression of QKI relative 2 MYB

M

to PLGGs without MYB-QKI MYB 0.5 1. 1. m (bottom). (

0 0 5 MYBtr da NIH3T3 cells allele. 1 MYB QKI promoter promoter i. 5 Fig.

234 and and MYB

y 33 MYB MYB - QKI Ex

` to to e a f on Qk ) Kaplan-Meier survival analysis of orthotopic SCID mice injected with with injected mice SCID orthotopic of analysis survival ) Kaplan-Meier ). ). - - - 56

. Luminescence represents ATP content. (

eGFP c MYB diseases in other relevance to shown have biological of a activation level 1.5-fold, approximately ( cells U87 in construct Q3E1- moter in the the from sequence enhancer proximal the Wecloned expression. MYB-QKI activate the NIH3T3 cells; MYB loop, feedback autoregulatory an possiblybybinding theto to contributes MYB-QKI that ( alone construct luciferase control promoterless the with MYB-QKI expressing stably cells in human glioma U87 activity gene ( human the with construct the activate tionally Proliferation n 7 10 We predicted that enhancers in the 3 the in enhancers that predicted We to func able was protein fusion MYB-QKI We whether examined = 5). Error bars, s.e.m. Expression of the signature in each sample sample each in signature the of Expression s.e.m. bars, Error = 5). 0 2 4 6 8 MYB - promoter in two additional cellular contexts (HEK293T and and (HEK293T contexts cellular additional two in promoter P MYB b luc =0.000

MYB 0 Luminescence relative MYBtr Days shLacZ eGFP MYB-QKI6 (shor MYB-QKI5 (shor eGFP reporter led reporter to even activity,higher again consistent with an -

to baseline (fold change) luc QKI 10 -

shQKI eGFP MYB 0 2 4 6 8 af luc - 3 1 MYB reporter as compared to U87 cells with the the with cells U87 to compared as reporter shLacZ MYBtr luc t er injection 3 Supplementary Fig. 6 Fig. Supplementary ). We observed significant induction of construct was higher than that of the the of that than higher was construct

A shLacZ MYB-QKI5 shQKI MYBtr MYB-QKI5 ` promoter when translocated, thereby further driving UTR (Q3E1) upstream of the human DVANCE ONLINE PUBLICATION ONLINE DVANCE 57 -

shQKI MYB-QKI5 luc t) t) MYB

shLacZ MYB-QKI6 * reporter construct. The baseline activity of the activity The baseline construct. reporter shQKI MYB-QKI6 ** promoter by creating a luciferase reporter reporter luciferase a creating by promoter 2 MYB-QKI6 MYB 6 i. 5 Fig. d

MYB 3 . Expression of MYB-QKI with the Q3E1- Tumor volume (cm ) 0 1 2 3 4 ** promoter upstream of the luciferase luciferase of the upstream promoter 1 promoter. MYB-QKI activated the the activated MYB-QKI promoter. c MY MYB-QKI6 (long) MYB-QKI5 (long) R 12 ), with activation increased by by increased activation with ), Days c etr c ) Expression of genes from the B ). o

Expression of genes 19 viral contr af ` UTR of of UTR associated with f t 26

Percent survival er injection

shQKI signature 10 50 200 300 400 0 0 10 33 0 0 0 ol 42 Fig. 5 QKI +MYB-QKI6 +MYB-QKI5 +MYBtr +eGFP

Nor 55 NATURE NATURE GE brain T e ime sur could aberrantly ) Hematoxylin ) Hematoxylin ma c P MYB ( ). This suggests MYB-QKI5 da MYB-QKI6 da ( n 10 l n < 0.000 = = NIH3T3 cells 0 * viv MYB 15 ( ( 10 Angiocentr n n MYB = =9) ed (d) ) promoter ) MYB gliom 1

10 N * - )

ETICS luc pro

a y 55 y 42 - 200 Qk

ic luc or

- -

© 2016 Nature America, Inc. All rights reserved. mNSCs mNSCs expressing MYB truncated or WeMYB-QKI. did with not observe injected mice from tumors intracranial five and MYB-QKI or mice injected with NIH3T3 cells overexpressing either truncated MYB ( trols) expressing truncated MYB or MYB-QKI fusion protein (15 vector con (five vector controls) and injections intracranial in 29 MYB mice of mNSCs or MYB truncated either overexpressing gliomas ( angiocentric tumor human in seen that OLIG2-positive to similar of pattern a subset cells, a and expression GFAP diffuse cytological atypia ( marked and mitoses frequent with features high-grade had they that in gliomas However, from human angiocentric tumors differed these Ink4a/ArfoverexpressionEGFRvIIIexample,(forin glioblastoma of adult models in seen histology the from distinct and glioma to of features tered growth similar those pattern, angiocentric clus a and vessels around growth enhanced of evidence some with resulted MYB-QKI6 in glioma formation or and tumor cells, infiltrating MYB-QKI5 overexpressing mNSCs with injections Intracranial ( xenografts flank as formationtumor to led MYB length full- of not but cells NIH3T3 in MYB-QKI6 and MYB-QKI5 both of ( cells NIH3T3 in overexpressed when growth anchorage-independent induced forms ( cantly proliferation increased rate as compared to expression of eGFP signifi a to led isoform either overexpressionof mNSCs, In cells. expressed human MYB-QKI5 and MYB-QKI6 in mNSCs and NIH3T3 human in diffuse gliomas ( observed that to similar pattern a cells, tumor of subset ( mice in injection intracranial after average, on d, 100 by formation glioma diffuse induced MYB truncated human expressing mNSCs ( flanks mouse into injected were cells when formation tumor induced MYB length overexpressioncells NIH3T3 ofhuman MYBtr to parison eGFP control ( rate the cell proliferation in exons com increased 1–9 variant) (short be to reported oncogenic been previously has MYB truncated of Expression MYB-QKI ( protein fusion the of loop in feedback the autoregulatory presence functions as a tumor-suppressor gene. suppression of the expression of (3) Hemizygous loss of loop. feedback autoregulatory an in resulting to bind to and activate the ability the exhibits and factor a transcription as functions oncogenic, is expressed is that protein fusion MYB-QKI The (2) activation. promoter of of H3K27ac-bound enhancers in the 3 QKI of the 3 MYB The mechanisms. three least at through oncogenesis Figure 8 NATURE NATURE GE tumors formation from any cells expressing vector control. These data P Supplementary Fig. 6c Fig. Supplementary Fig. 6e Fig. < 0.0001; 0.0001; < QKI In total, we established flank injections in 15 mice of NIH3T3 cells cells in of 15 mice NIH3T3 injections flank In we total, established stably we oncogenic, are fusions MYB-QKI whether test To . (1) This results in proximal translocation translocation proximal in results This . (1) MYB and and to the the to Supplementary Fig. 7b Fig. Supplementary `

portion of of portion , - f MYB QKI QKI ). These tumors expressed OLIG2 and GFAP protein in a in protein GFAP and OLIG2 expressed tumors These ). 10–

N fusion Fig. Fig. 6 MYB , resulting in hemizygous deletion deletion hemizygous in , resulting rearrangement disrupts both both disrupts rearrangement ETICS - 1 QKI 2 . In mNSCs, overexpression of truncated MYB from from MYB truncated of overexpression mNSCs, In . Fig. 6 Fig. promoter, resulting in in resulting promoter, Supplementary Fig. 6c Fig. Supplementary rearrangement contributes to contributes rearrangement MYB

c Supplementary Fig. 7a Fig. Supplementary

protein and and Fig. 6 Fig. ADVANCE ONLINE PUBLICATION b QKI Fig. 5 Fig. and the 5 the and and Supplementary Fig. 6b Supplementary ). MYB results in results Fig. Fig. 6 e ). Immunohistochemical analysis showed showed analysis Immunohistochemical ).

is c Supplementary Fig. 6b Fig. Supplementary ).

promoter, promoter, oncogenic QKI ). We observed flank tumors in all 15 15 all in tumors flank We observed ). a ` portion of portion and and , which , which ` region region Supplementary Fig. 6b Supplementary MYB ).

). In vivo In exons1–15 ). Similarly, both iso - QKI fusion protein protein fusion QKI 1 Au , overexpression overexpression , to

). Furthermore, Furthermore, ). H3K27ac r egulat −/ Chr MY but not full- not but activ . mNSCs) 6 B or 2 pr Fig. 6 Fig. ation of y f ), ), and in omot eedbac No H3K27acpeaks er Tr d 2 uncat k loop 7 ). ). - - - - - .

MY oncogenic MYB-QKI ed B MYB brain human normal ( to relative gliomas angiocentric in enriched ( pression correlatedwith most expressionwas whose genes 50 the QKI contain a miRNA regulatory element (MRE) for miR-717 (ref. (ref. miR-717 for (MRE) element regulatory miRNA a contain ( wild-type of suppression of with miRNAs ten expression the regulate to (miRNAs) microRNAs reported been previously has QKI instead cells to expressing relative control shRNA to genes 309 of expression differential to led tive functional effects. and overexpression MYB-QKI that in on 5 day ( increase proliferation mild a observe did we although eGFP, expressing cells in suppression of d 3 within We proliferation MYB. increased observe not did truncated or eGFP overexpressing cells to compared as of suppression of degree lower or similar a despite protein, fusion MYB-QKI in overexpressing cells was observed effect ( suppression of wild-type of suppression MYB-QKI6, or MYB-QKI5 MYB, truncated human of preexisting human MYB-QKI expression. In mNSCs overexpressing liferation of mNSCs, with the greatest increase observed in the context (shRNAs) that targeted the first four exons of of expression reduced QKI We were interested in understanding how disruption of of disruption how understanding in interested We were MYB protein fusion or ( MYB MYB-QKI ing truncated express in cells of formation tumor enrichment a significant represent ( QKI with gliomas angiocentric of analysis RNA-seq Exon-specific rangement. harbor that tumors in oncogenicity to contribute MYB Expr Fig. Fig. 7 P Supplementary Table 7 Table Supplementary < 0.0001; 0.0001; < Taken together, our which through data mechanisms three suggest with consistent effects molecular exhibit gliomas Angiocentric Suppression of of Suppression wild-type suppressing Indeed, ession of as compared to PLGGs that harbored harbored that PLGGs to compared as rearrangement might contribute to tumor formation through through formation tumor to contribute might rearrangement suppression. In mNSCs, we defined a signature consisting of of consisting signature a defined we mNSCs, In suppression. MYB - - QKI QKI a and Supplementary Fig. 8c Fig. Supplementary - Qk rearrangement contributes to oncogenicity ( oncogenicity to contributes rearrangement

QKI QKI rearrangement Supplementary Note Supplementary Fig. 7 Fig.

was sufficient to increase proliferation within only 3 d only within proliferation increase to sufficient was ex ons 5–8 MY rearrangement ( rearrangement Fig. 7 Fig. B c Qk pr Loss of ). omot by shRNA in mNSCs expressing MYB-QKI6 MYB-QKI6 expressing mNSCs in shRNA by QKI b Supplementary Fig. 8b Fig. Supplementary 28 and er activ 3 ,
2 ). Isoform 7 of mouse of 7 Isoform ).

MY 9 , a tumor-suppressor gene. tumor-suppressor a ,

, and we also observed upregulation of of upregulation observed also we and , disrupts lacZ 3 B Supplementary Fig. 8a Fig. Supplementary ation n ( = 4) showed reduced expression of = expression 4) reduced showed ). ). These data suggest that the ). This signature was significantly significantly was signature This ). q RNA biniding < 0.25; 0.25; < Qk QKI

QKI using short hairpin RNAs RNAs hairpin short using suppression exert coopera exert suppression enhancer translocation H3K27ac H3K27ac peakspr tumor suppr Disr ,

Supplementary Table 6 Supplementary BRAF a uption of

Qk Qk tumor q23.3 MYB QKI ). These data suggest suggest data These ). led to increased pro increased to led , including miR-717 miR-717 including , P alterations ( Qk essor ARTICLES < 0.0001). < QK Qk

MYB is predicted to to predicted is suppressor I ). The greatest greatest The ). esent in these cells cells these in Fig. Fig. q25.3 - QKI QKI 8 Qk QKI ). First, First, ). might might n MYB rear 3 sup = 5) 5) = 0 ). ). ). 7 ------

© 2016 Nature America, Inc. All rights reserved. ing cooperation between its loss and between of expression ing cooperation confirming overexpression, undefined. remain of role functional the tern, pat expression this underlying mechanism the although expression, Like CDK7 (ref. bromodomainBET as suchmechanisms, indirect through raises fusion the of also effects the inhibiting elements therapeutically of possibility the enhancer H3K27ac-bound with MYB-QKI transcriptional MYB-QKI as such targets challenging, be to likely is MYB target transformation. for sufficient borderline be may gliomas in Ink4a/Arf EGFRvIII of overexpression example, (for models glioma grade low The high- to relative attempts) 29 histologies. in tumors (five mNSCs for all penetrance across rare are gliomas adult or pediatric of low-grade models Successful index. low mitotic a very and exhibit tumors I grade WHO are gliomas Angiocentric tumors. intracranial ependymomas. or gliomas diffuse IDH-mutant as such with higher potential for recurrence or that require treatment, further tumors from gliomas angiocentric distinguishing in aid could This glioma. angiocentric for diagnostic considered be should fusion this glioma as a entity.separate biological We propose that the presence of with this histology translocation supports pathological the classification of of angiocentric association tight The disease. this of diagnosis centric glioma. This has important implications for the treatment and gene. tumor-suppressor a of loss functional ous enhancer elements regulating fusion gene expression and simultane of translocation proximal loop, feedback autoregulatory an in pates of expression protein fusion an oncogenic that activated then partici aberrant in the resulting of genes two translocation driver of a single It glioma. centric example, the to represents first also our knowledge, describe We DISCUSSION manner. cooperative a in formation tumor to both of disruptions Thus, gene. of in suppression results ing in an autoregulatory feedback loop. Third, hemizygous loss of the to to and activate bind ability the exhibits and factor as a transcription functions is that oncogenic, is expressed in 3 the in enhancers H3K27ac-bound of translocation proximal in results alteration the ARTICLES 8 brain. This hypothesis is supported by the essential role of QKI in in QKI of role essential the by supported is hypothesis This brain. of loss complete that be may gliomas angiocentric in haploinsufficiency the for tion glioblastomasdemonstrate gene.biallelicexplanathisOneofloss cooperativity of between mechanisms evaluate to required is investigation sion of genes implicated in cancer transition via regulation of circular RNAs alteration of splicing events the including cancer, in QKI for roles of diversity a indicate studies

We observed an additive effect for for effect additive an We observed harbor carcinomas cystic Adenoid to inhibitors directly of molecule the development small Although generate reproducibly to sufficient was expression MYB-QKI found We Angiocentric gliomas exhibit haploinsufficiency for haploinsufficiency exhibit gliomas Angiocentric MYB MYB promoter activation. Second, the MYB-QKI fusion protein protein fusion MYB-QKI the Second, activation. promoter QKI - −/ QKI 3 MYB MYB 3 mNSCs) suppression and MYB-QKI expression. MYB-QKI and suppression ) inhibition. ) , MYB KIT - - QKI QKI QKI ` region of or - exerts negative selection in the developing developing the in selection negative exerts NFIB 2 rearrangement to be a defining event in angio in event defining a be to rearrangement as a new recurrent diagnostic fusion in angio in fusion diagnostic recurrent new a as 7 suggests that genetic drivers of low-grade low-grade of drivers genetic that suggests CDK6 QKI NFIB 3 7 fusion also results in high levels of MYB levels in high results also fusion QKI , which functions as , a functions which tumor-suppressor and a role in epithelial-to-mesenchymal can be targeted. The association of of association The targeted. be can QKI and the oncogenicity of oncogenicity and the MYB as a tumor suppressor and suggest and suppressor tumor a as 3 9 and miRNA processing miRNA and to the Qk and MYB suppression and MYB-QKI MYB-QKI and suppression 3 8 . QKI regulates the expres MYB QKI - MYB NFIB appear to contribute contribute to appear promoter, resulting resulting promoter, MYB promoter, result alterations QKI - QKI MYB 3 0 , whereas , whereas . . Further . . Recent 31 - NFIB , 34– 3 QKI 2 or or 3 6 ------.

Note: Any Supplementary Information and Source Data files are available in the accession study under (dbGaP) Phenotypes and deposited Genotypes of database been the in has ChIP-seq) and RNA-seq sequencing, genome sequencereportedpaperthis(whole-exome in sequencing, whole- series under (GEO) Omnibus Expression Gene codes. Accession the in available are pape the of version references associated any and Methods M com/Bradn http URLs. tumors. pediatric of number large a to utes to through multiple oncogenicity may mechanisms be applicable alterations driver glioblastomas. to compared as gliomas angiocentric of nature lower-grade the for oligodendrocytic differentiation. Haploinsufficiency may also account Y.J.K., D.K., B.R.P., A.G.-Y., P.M.H., A.F., H.M., A.A.T., S. Seepo, M.D., P.V.H., D.A.H., J.J.P., A.J.W., P.B.S., J.W., S.E.S., R.Z., L.U., R.O’R., W.J.G., K.P.,G.B., H.J.H., S.H.R., Y.Z., N.C., A.S., K.B., M. R.E.H., Santi, A.M.B., M. S.M., Scagnet, P.B., P.J.,L.A.R., G.B., K.L.L., R.B. and research.designed A.C.R. P.B., P.J.,L.A.R., PLGGs for generous their contributions to research. this (D.A.H.-K.). Foundation Astrocytoma and Stephen Grand Philanthropic Fund (D.A.H.-K.) and the Pediatric Low-Grade Academy of (A.K.), NINDS Sciences grant (D.A.H.-K.),1R01NS091620 the Nancy grant KTIA_13_NAP-A-V/3, the Janos Bolyai Scholarship of the Hungarian Thea’s Star of Hope and (A.C.R. A.J.W.), the Hungarian Brain Research Program, the Pediatric(S.H.R.), K08NS087118 Brain Tumor Foundation (R.B. and P.B.), (A.J.W.), National Institute of Neurological Disorders and Stroke (NINDS) grant (A.J.W. and Foundationthe A.C.R.), Rally forCancer Research Childhood Hyundai Scholar Grant (A.J.W.), the NecessitiesBear Pediatric Cancer Foundation Martin (J.G.), the Damon Runyon-Sohn Pediatric Fellowship Award (A.J.W.), a Soldati Foundation (G.B.), the Philippe Foundation (G.B.), Fondation Etoile de R.B. and S. Santagata), the Sontag Foundation (K.L.L. and R.B.),Sunflowers for Life theFund for Nuovo-Pediatric Brain and Spinal Cancer Research (P.B., Accelerate Research and Collaboration (SPARC) grant (A.G.), the Jared Branfman Fund the Andrysiak for LGG (M.W.K.), a Broad Institute Projects Scientific to Association the Team(L.A.R.), Jack Foundation (P.B., M.W.K., R.B. and L.G.), K.L.L. and R.B.), St. Baldrick’s Foundation (P.B.), the American Brain Tumor Foundation (K.L.L.), US National Institutes of Health grant (C.D.S.,PO1CA142536 and Shop Pediatric Brain Tumor Program (P.B. and M.W.K.), the Path to Cure the Children’s(A.C.R.), Brain Tumor Foundation and (A.C.R. A.J.W.), the Stop R01NS085336 (A.C.R., A.J.W., P.B.S. and M. Santi), Voices Against Brain Cancer R.B., M.W.K., L.G., C.D.S. and A.C.R.), US National Institutes of Health grant Foundation Pediatric Low-Grade Astrocytoma Foundation (P.B., K.L.L., laboratories for useful discussions. technical assistance with FISH, and members of the Ligon, Resnick and Beroukhim Brigham and Women’s Hospital, H. Homer (Brigham and Women’s Hospital) for analysis, the Neuro-Histology laboratories of Boston Children’s Hospital and Firehose Team at the Broad Institute for assistance with genomic sequencing and Pittsburgh for sample contribution, members of the Genomic Platform and Children’s Hospital, Seattle Children’s Hospital and Children’s Hospital of Consortium, including additional participating sites: Ann and Lurie Robert Low-Grade Astrocytoma Consortium and the Children’s Brain Tumor Tissue We thank and acknowledge the Dana-Farber Harvard Cancer Center/Pediatric pape of the online version AUTHOR CONTRIBUTIONS AUTHOR A CKNOWLEDGMENTS Finally, we thank and acknowledge the many children and families affected by We acknowledge the following funding sources: A Kids’ Brain Tumor Cure ET Pediatric tumors are characterized by simple genomes with single single with genomes simple by characterized are tumors Pediatric ://broadinstitute.gi H SAMtools, ODS phs001054.v1.p erLab/pipeline/wiki/ A DVANCE ONLINE PUBLICATION ONLINE DVANCE 40 Microarray data have been accessioned with the the with accessioned been have data Microarray , r 4 . h r 1 . . Our findings that one rearrangement contrib one that rearrangement . findings Our ttp://www.samtools.s thub.io/picard 1 . bamliquidato / ; ; bamliquidator, ourceforge.net r .

NATURE NATURE GE GSE7579 http s://github. / ; Picard, Picard, ; N 6 online ETICS . The The . -

© 2016 Nature America, Inc. All rights reserved. 1 22 Hospital, University of Florence, Florence, Italy. Philadelphia, Pennsylvania, USA. Philadelphia, Philadelphia, Pennsylvania, USA. Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. Sainte-Anne, Université Paris V Descartes, Paris, France. Boston, Massachusetts, USA. Massachusetts, USA. Therapeutiques du Cancer’, Gustave Roussy, Université Paris XI Sud, Villejuif, France. Cancerologie de l’Enfant et de l’Adolescent et Unité Mixte de Recherche du Centre National de la Recherche Scientifique 8203 ‘Vectorologie et Nouvelles Graduate Group, Gene Therapy and Vaccines Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. USA. Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. Boston, Massachusetts, USA. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints pape of the version The authors declare competing financial interests: details are available in the edited and approved the manuscript. the manuscript. K.L.L., the R.B. study.and supervised A.C.R. All authors reviewed, algorithms and/or samples. P.B., P.J.,L.A.R., G.B., K.L.L., R.B. and wroteA.C.R. J.J.P., F.J.R., S. Santagata, N.J., A.G., J.G., A.H.L. and L.G. contributed new reagents, A.K., L.B., P.C.B., C.E., M. Santi, A.M.B., M. S.M., D.A.H.,Scagnet, D.A.H.-K., K.L.L. and R.B. analyzed the data. J.W., P.V., M.P., D.C.B., C.G., S.P., C.H., U.T., P.B.S., A.F.,R.Z., S.E.S., W.J.G., S. S.H.R., Santagata, A.G., J.E.B., A.H.L., A.C.R., completedA.C.R. the research. P.B., P.J.,L.A.R., G.B., J.W., H.J.H., Y.Z., A.J.W., S. Santagata, C.D.S., J.E.B., N.J., A.G., J.G., A.H.L., L.G., M.W.K., K.L.L., R.B. and D.C.B., S.P., C.H., U.T., A.K., L.B., P.C.B., C.E., F.J.R., D.A.H., D.A.H.-K., NATURE NATURE GE COMPETING FINANCIAL INTERESTS Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.

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Department Department of Pathology, Brigham and Women’s Hospital, Boston, hn A.-J. Chen, Wang, Y., Vogel, G., Yu, Z. & Richard, S. The QKI-5 and QKI-6 RNA binding proteins R.M. Bachoo, S. Menzel, the in elements DNA of encyclopedia integrated An Consortium. Project ENCODE P.A. Northcott, Mansour, M.R. R. Gao, (QKI-5) protein I-5 quaking The K. Artzt, & Tee,Tonissen,R. K., L., Zhou, J., Wu, T.I.Zack, Bian, Y. Y.Zhao, D. Yin, eoki, R. Beroukhim, Kieran, M.W. B.D. Crompton, W. Lu, S.J. Conn, M. Danan-Gotthold, P.J. Stephens, M. Persson, T.M. D’Alfonso, E. Chipumuro, J.E. Delmore, P. Bandopadhayay, S. Ji, stabilization of specific miRNA. specific of stabilization (2013). regulate the expression of microRNA 7 in glial cells. axis. astrocyte to cell stem neural the along transformation and differentiation terminal governing mechanisms humans. in counts (2007). monocyte 3624–3626 and platelet, erythrocyte, influences genome. human medulloblastoma. element. intergenic noncoding a of (2014). 12528–12542 targets. therapeutic MYB-independent and MYB-dependent parallel the and cytoplasm. nucleus the between shuttles and signal localization nuclear novel a has 45 prognosis. cancer in protein. prognostic and diagnostic 7 microarray. DNA polymorphism nucleotide single by multiforme 20007–20012 (2007). 20007–20012 glioma. to application and methodology cancer: tumors. rhabdoid pediatric Discov. SOX2. of repression Cell (2015). tumors. solid human across events splicing Invest. Clin. J. neck. and head and 106 breast the of carcinomas in features. basaloid with variant solid the to paid Pathol. Hum. focus special with breast the cancer. MYCN-driven in transcription c-Myc. medulloblastoma. cancer. colorectal of development the and signalling Department Department of Pathology and Laboratory Medicine, Children’s Hospital of , 665–677 (2009). 665–677 , , 1134–1140 (2013). 1134–1140 ,

2 , 18740–18744 (2009). 18740–18744 , 160 Dana-Farber/Boston Children’sDana-Farber/Boston Cancer and Blood Disorders Center, 23

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20

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5 9 , , ,

© 2016 Nature America, Inc. All rights reserved. Women’s Hospital, Boston, Massachusetts, USA. USA. should be addressed to K.L.L. ( Massachusetts, Boston, Hospital, Women’s 48 Philadelphia, Pennsylvania, USA. Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. Massachusetts, USA. USA. Canada. Quebec, Canada. Boston, Massachusetts, USA. USA. Radiation Oncology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston Children’s Hospital, Boston, Massachusetts, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco School of Medicine, San Francisco, California, USA. USA. Medicine, San Francisco, California, USA. 30 Center, Washington, DC, USA. Hungary. The Hospital for Sick Children, Toronto, Ontario, Canada. Université Paris V Descartes, Paris, France. ARTICLES 1 0 Center for Data-Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. Department of Pathology, Children’s National Medical Center, Washington, DC, USA.

42 36 33 Department of Neurosurgery, Boston Children’s Hospital, Boston, Massachusetts, USA. Department of Pathology, University of California San Francisco, San Francisco, California, USA. Department of Pediatrics, University of California San Francisco School of Medicine, San Francisco, California, USA. 41 27 Brigham Brigham and Women’s Hospital Department of Pathology, Center for Advanced Molecular Diagnostics, Division of Boston, Cytogenetics, Massachusetts, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 39 Department of Human Genetics, McGill University, Montreal, Quebec, Canada. 44 Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 38 29 [email protected] Division Division of Experimental Medicine, Montreal Children’s Hospital, McGill University and McGill University Health Centre, Montreal, Center Center for Neuroscience and Behavioral Medicine, Brain Tumor Institute, Children’s National Medical Center, Washington, DC, USA. 47 Department Department of Biomedical and Health Informatics, Children’s Hospital of USA. Pennsylvania, Philadelphia, Philadelphia, 32 24 Department Department of Neurological Surgery, University of California San Francisco School of Medicine, San Francisco, California, Division of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada. 50 These authors contributed equally to this work. 26 Department Department of Neurosurgery, Medical and Health Science Center, University of Debrecen, Debrecen, ), A.C.R. ( 46 [email protected] Department Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, 31 Department Department of Neurology, University of California San Francisco School of ) or R.B. ( 43 Department Department of Neurosurgery, Harvard Medical School, Boston, 51 40 These These authors jointly supervised this work. Correspondence Department Department of Pediatrics, McGill University, Montreal, Quebec, A 37 [email protected] DVANCE ONLINE PUBLICATION ONLINE DVANCE Department Department of Medicine, Harvard Medical School, 28 Brain Brain Tumor Institute, Children’s National Medical 45 Center Center for Childhood Cancer Research, 49 Department Department of Medicine, Brigham and 34 25 Department Department of Radiation Oncology, Division Division of Haematology/Oncology, 35

). Department Department of NATURE NATURE GE N ETICS © 2016 Nature America, Inc. All rights reserved. removed by filtering against the ESP and Exome Aggregation Consortium Consortium Aggregation Exome and ESP the against filtering by removed were SNPs germline Likely Oncotator. with genes to annotated and samples GATK. with achieved was deletions and insertions around realignment and local recalibration score quality Base tools. Picard with cates and indexed parameters -q 5 -l 32 -k 2 -t 4 -o 1. data Aligned were sorted, marked for dupli set. bait Mb) (44 v2 Exon All Human Agilent the with performed was capture hybrid Exome analysis. for multiplexed TruSeq) (Illumina adaptors barcoded to specific ligation and XP beads) AMPure (Agencourt by of two rounds selection followed size tion, sonica Covaris by prepared were size insert average 250-bp a with Libraries kit. and DNA Tissue Qiagen the Blood using DNA extracted was sequencing. whole-genome by identified alterations driver confirm to used were samples formalin-fixed, paraffin-embedded samples (without matched controls). These sequencing. Whole-exome 5). = width minimum ment the copy number data seg ( to used was segmentation binary circular and variations, number copy germline known of cleaned were data number copy content–normalized GC embedded samples and array CGH was performed as previously described CGH. Array inspection. by visual assessed also were files BAM scripts. tran fusion of detection the for and metrics quality and levels expression exon levels, expression gene determine to Firehose within PRADA Weused analysis) data sequencing RNA for (pipeline PRADA using preprocessed and genome reference hg19 the to aligned were reads paired-end Raw rithm. SamToFastq Picard algo the using files fastq to transformed were files BAM sets data published from downloaded were files RNA-seq read. barcode index an 8-base including run paired-end a 76-bp with instruments, performed according to the manufacturer’s protocols using HiSeq 2000 or 2500 Illumina TruSeq protocol. Flow cell cluster amplification and weresequencing a using non-strand-specific performed was construction library Qiagen), kit, RNA sequencing and analysis pipeline. Firehose within performed were analyses All inspection. visual and Rearrangements and breakpoints were identified using dRanger, BreakPointer 2.0) (version (IGV) Viewer Genomics Integrative the in inspection visual and point mutations and short indels were using called MuTect alterations number copy recurrent identify to used was 2.0 (GATK) Toolkit Analysis Genome the using cycle machine and context dinucleotide were to score assignments recalibrated control for due biases to flow cell, lane, and normalized coordinates, by and cleaned, duplicates were marked sorted using SAMtools and Picard. quality Base were Reads 1. --o 2 --k 32 --l 5 --q (BWA) Aligner Burrows-Wheeler the using 37) (Build hg19 PLGG published Illumina recently accessed were an from sets data on files Sequencing sequencing instrument. paired-end 2000 for HiSeq prepared were fragmented, randomly libraries was and DNA Institute. Hospital Children’s Broad the at and Institute Philadelphia Genomics of Beijing Consortium the at Astrocytoma sequenced were Low-Grade Dana-Farber the Center–Pediatric and Cancer Philadelphia Harvard of Hospital CBTTC–Children’s from processing. and sequencing Whole-genome consent. informed of waiver IRB specific with samples deidentified as analyzed were or samples of collection the before consent informed provided patients all and was obtained, institutions all from approval IRB Philadelphia. of Hospital Children’s and Institute Broad the Children’s Hospital, Boston Institute, Cancer Farber (IACUC) of Dana- the committees research animal and/or (IRB) board review statement. Ethics ONLINE doi: (ExAC) databases. Mutations were called with MuTect, filtered against a panel of normal normal of panel a against filtered MuTect, with called were Mutations BWA with genome to reference hg19 the were data aligned Sequence using 10.1038/ng.3500 4 4 . Copy number alterations were evaluated using SegSeq using evaluated were alterations number Copy .

MET DNA was extracted from archival formalin-fixed, paraffin- 4 9 was applied to detect significantly recurrent mutations. mutations. recurrent significantly detect to applied was H Ethics approval was granted by relevant human institutional ODS 2 , 5 . Read pairs were aligned to reference genome genome reference to aligned were pairs Read . Whole-exome sequencing was performed from from performed was sequencing Whole-exome A = 0.001, undo.splits = sdundo, undo.s.d. = 1.5, = undo.s.d. sdundo, undo.splits = 0.001, Following RNA extraction (RNeasy (RNeasy RNA extraction Following PLGGs and normal controls controls normal and PLGGs 4 7 and IndelLocator 20 4 3 , 42 with options options with 2 , , 4 5 4 4 6 . RNA-seq RNA-seq . 5 8 . Somatic Somatic . 5 . GISTIC GISTIC . . MutSig MutSig . 1 . 1 , 5 5 5 2 3 0 - - - - - . .

vector was used to clone the pLightSwitch_Prom the in site MluI-BglII promoter.The a without (RenSP) contains a multiple-cloning site upstream of a 8 Table MYB constructs. enhancer containing constructs reporter the Generation of quantified. were pixels 500 than greater area an with colonies and software, ImageJ using analyzed were Images acquired. were images and in 96-well plates (in colonies Cell triplicate). were allowed to form for 2 weeks, DBS and DMEM with agar 0.7% in plated were control vector retroviral and proteins QKI full-length and MYB full-length MYB, MYB-QKI5, truncated the MYB-QKI6, of each expressing cells NIH3T3 modifications. following performed across 10,570 tumor samples from 31 lineages from TCGA, as as TCGA, from lineages 31 from samples tumor 10,570 across performed of Analysis MYB levels. from pole of the occipital left a adult normal brain autopsy were to used assess stain, and coverslips were mounted with Permount (Fisher Scientific). Sections Mayer’s hematoxylin using performed was nuclei for Counterstaining tion). and dilution) GFAP1:500 dilu AB9610; 1:10,000 OLIG2 (Chemicon, (Sigma; tissue), mouse for dilution) 1:1,000 (A304-135A; Laboratories Bethyl tissue, for human dilution) 1:100 (ab45150; (Abcam to MYB for antibodies retrieval antigen for used were 6.0) (pH buffer citrate sodium mM 10 and Heat tions. on protocols 5- to standard according performed Immunohistochemistry. control. a as co-hybridized 6 was of chromosome region centromeric the to mapping (Invitrogen) probe 6q23.3. (3 RP11-170P19 (5 RP11-63K22 probes 5- MYB criteria. 2007 WHO using S.H.R.) or Santagata S. (K.L.L., neuropathologist board-certified a by classified and reviewed trally cen were published previously not Samples data. published previously to assignment. Histological ent growth of NIH3T3 cells was assayed as previously described previously as assayed was cells NIH3T3 of growth ent Soft agar colony formation assays and quantification. dilution). 1:1,000 A300-183A; Laboratories, to QKI (Bethyl antibody 25; 1:5,000 dilution), antibody to MYB (Abcam, ab45150; 1:5,000 dilution) and horseradish peroxidase (HRP)-conjugated antibody to Myc (Invitrogen, R951- expression of Myc-tagged proteins was confirmed via Stable immunoblotinfection. after h analysis 48 withcommenced was puromycin with selection and h, 6 for medium retrovirus-containing with infected were cells NIH3T3 tocols. were to BioLabs) used to according generate retrovirus the manufacturer’s pro Puro retroviral vector (Cell Biolabs). Platinum-E retroviral packaging cells (Cell pMXs- N-Myc–tagged Gateway-compatible into a subcloned were constructs QKI and MYB full-length and MYB, truncated MYB-QKI6, and MYB-QKI5 as chased Gateway clones entry DNAfrom PlasmID/DF/HCC Resource Core. MYB-QKI fusions as templates. Full-length MYB and QKI constructs were pur entry clones. Truncated were MYB constructs generated via PCR mutagenesis using Gateway-compatible as synthesized were constructs MYB-QKI6 and lines. stable NIH3T3 of generation and construction Vector ANOVA and loaded from the GTEx Consortium down were tissues autopsy normal from obtained RNA-seq from levels sion the of Atlas BRAINSPAN developing the human brain from and processed as previously accessed described were samples brain pediatric tissues. normal in expression gene of Analysis described previously M m formalin-fixed, paraffin-embedded tissue sections and Homebrew Homebrew and sections tissue paraffin-embedded formalin-fixed, m promoter activity, the human FISH analysis. ) was cloned into the pLightSwitch_Prom vector (Active Motif) that that Motif) (Active vector pLightSwitch_Prom the into cloned was ) MYB QKI status was assessed in 50 tumor nuclei per sample. A A sample. per nuclei tumor 50 in assessed was status t tests. tests. ` alterations in TCGA samples. to P 2 MYB FISH was performed as previously described 0 values <0.05 were considered to be significant. be to considered were <0.05 values To assess the effect of candidate enhancer regions on on regions enhancer candidate of effect the Toassess . ` to Diaminobenzidine (DAB), staining was bright-field Diaminobenzidine ; directly labeled in SpectrumGreen) that map to to map that SpectrumGreen) in labeled directly ; Histological subtype assignments were according according were assignments subtype Histological MYB MYB promoter sequence ( ; directly labeled in SpectrumOrange) and and SpectrumOrange) in labeled directly ; MYB 1 4 . Expression levels were using compared levels . Expression promoter ( sequence Renilla M GISTIC 2.0 analyses were were analyses 2.0 GISTIC RNA-seq data for normal normal for data RNA-seq m paraffin-embedded sec Supplementary Table Supplementary 9 luciferase reporter gene reporter luciferase Anchorage-independ MYB NATURE NATURE GE promoter and and promoter Supplementary Supplementary 5 5 . MYB MYB-QKI5 MYB-QKI5 5 CEP6 6 with the the with 5 N expres 4 using using ETICS aqua ), ------

© 2016 Nature America, Inc. All rights reserved. 96-well plates, with five replicates. Cell viability was measured by assessing ATP assays. proliferation Cell infection. after h 48 assays tion (931 protocol spin a using infection underwent Target mNSCs concentrated. and (Invitrogen, Lipofectamine 56532). Lentivirus-containing supernatant using was collected 48 VSVG h after transfection and PSPAX2 encoding plasmids (10 shRNA each encoding vectors with cells HEK293T ( exonsof for mouse shRNAs specific encoding Qk infection. after h 48 commenced (0.5 selection Puromycin polybrene). no with °C 30 at min 120 for ultrafiltration). Target mNSCs underwent infection using a spin protocol (931 supernatant was 48 collected h after and pooled transfection, concentrated (by mids encoding PSPAX2 and VSVG using Lipofectamine. Lentivirus-containing from D. Root (Broad Institute); Addgene plasmid 41392) with plas packaging 10 with transfected were Overexpressionof transcripts in mouse neural stem cells. Cell). Stem (07980, heparin and Millipore) GF003, (FGF; factor growth fibroblast Cell), Stem 02653, (EGF; factor growth epidermal (Gibco), DMEM (Gibco) and neural stem cell medium (Gibco) supplemented with B27 ously described C57BL/6 wild-type E14.5 mouse embryos (purchased from Taconic) as previ cells.stemneural Generation of infection. mycoplasma for months) 3 every least (at tested routinely were lines cell All reauthenticated. not were and ACTT from Cell lines. Dual-Luciferase the using transfection manufacturer’s the to protocol. according (Promega) System Assay after Reporter h 24 quantified was activity Luciferase 2000. Lipofectamine via controls or fusions indicated with along pRL served as an internal control in all assays. The pRL The HindIII. and XhoI with digested (Promega) vector into pGL4.10[luc2] ligated was cleotide and forward ( reverse mim-1 Double-stranded oligonucleotides were generated by annealing primers mim-1 the in present is which PyAAC(G/T)G, sequence consensus (MRE) element MYB recognition for c-MYB were generated. The reporter construct was designed using the core Luciferase reporter containing constructs a consensus DNA-binding sequence mim-1 protocol. manufacturer’s the to according Motif) (Active Reagent Assay Luciferase LightSwitch using transfection after ing MYB-QKI using PolyFect (Qiagen). Luciferase activity was quantified 24 h cells 2000 via (Invitrogen) Lipofectamine or into NIH3T3 lines stably express 3000 ( Lipofectamine U87 using MYB-QKI) the expressing stably into line U87 a (or line glioma transfected were enhancers with together or alone promoter the containing constructs reporter luciferase The assays. all for control LightSwitch promoter, gene housekeeping a with vector reporter A control. negative a as used was gene reporter luciferase RenSP the of upstream cloned fragment genome human the non-repetitive from and non-genic non-conserved, 1-kb a Motif) (Active containing 1 Control Promoter Random as LightSwitch above. constructs described reporter the into cloned and Invitrogen by synthesized were and chr. 6: 163,921,548–163,921,972) regions chr. (hg18, 6: 163,920,360–163,920,809 enhancer candidate clone to used of the upstream further was site MluI the and NATURE NATURE GE Means (Promega). CellTiter-Glo using content upeetr Tbe 9 Table Supplementary shRNA experiments and proliferation assays. proliferation and experiments shRNA g Renilla for 120 min at 30 °C with no polybrene). Cells were placed into prolifera into placed were Cells polybrene). no with °C 30 at min 120 for n eotr osrc gnrto ad Y tasciain assays. transactivation MYB and generation construct reporter = 5) or with MYB-QKI or vector control cotransfected into HEK293T Qk NIH3T3, HEK293T and U87 MG cell lines were obtained directly directly obtained were lines cell MG U87 and HEK293T NIH3T3, vector (at a ratio of 30:1) were cotransfected into HEK293T cells cells HEK293T into cotransfected were 30:1) of ratio a (at vector , and control shLacZ were obtained from the RNAi Consortium Consortium RNAi the from obtained were shLacZ control and , N ETICS 5 7 MYB mim-1 . mNSCs were maintained in culture medium with 1:1 ratio of promoter. The human gene promoter, a previously described MYB target MYB described previously a promoter, gene Renilla M Cells were plated at a density of 1,000 cells/well in in cells/well 1,000 of density a at plated were Cells ). Lentivirus was produced by transfection of of transfection by produced was Lentivirus ). g of pLEX307 lentiviral expression vectors (a gift gift (a vectors expression lentiviral pLEX307 of g Supplementary Table Supplementary 9 ACTB luciferase reporter vector (Promega, E2261) E2261) (Promega, vector reporter luciferase Promoter Control, was used as a positive positive a as used was Control, Promoter Embryonic mNSCs were derived from from derived were mNSCs Embryonic Qk , targeting sequences in the first four first in the sequences , targeting QKI mim-1 o 3 s.e.m. were calculated. were s.e.m. ` Lentiviral vectors (pLKO) vectors Lentiviral UTR enhancer sequences sequences UTR enhancer ). The annealed ). oligonu The annealed reporter construct and and construct reporter M g) with packaging packaging with g) HEK293T cells cells HEK293T M g/ml) g/ml) MYB 8 g - - - - - .

the antibody and concentrations that produced the highest signal-to-noise signal-to-noise highest the produced that concentrations and antibody the Antibody optimization and ChIP-seq analysis. of settings). (default module GenePattern ClassNeighbors the using defined was signature MYB-QKI The Gene sets with nominal (MSigDB). sets gene (CP) C2 the using performed was GSEA expression. in with Genes analysisselection normalized RMA 2.0 were Gene files Mouse CEL microarrays. Affymetrix ST using assayed were profiles expression Gene QKI. or truncated MYB-QKI6 one of ing eGFP, MYB-QKI5, MYB, truncated express mNSCs of pools generated independently three from extracted was MYB-QKI. expressing cells stem neural of analysis expression Gene the using were analyzed in as previously described RT-PCR was performed Real-time RNA-to-cDNA Biosystems). (Applied kits 1 from synthesized was cDNA (Qiagen). kit RNA extraction and real-time RT-PCR. dilution). 1:2,000 Biotechnology; Cruz Santa (sc-1615, actin and Abcam; 1:500 dilution), QKI (A300-183A, Bethyl Laboratories; 1:1,000 dilution) described previously Immunoblotting. by Active Motif as recently described as recently Motif by Active brain normal from maps matin IGV.with chro data for visualized ChIP-seq H3K27ac of published BED files as a heat map. Peaks and were alignments converted to TDFs by IGV tools and plotted and genome the across experiment each in observed value largest the 50 bins, and read density was calculated in each bin. Density was normalized to enriched H3K27ac peaks were extended 5 kb in each direction and divided into ized to read density in units of RPM/bp. To overlap,genome-wide calculate all 200 were bp Reads extended version 1.1.0. and normal bamliquidator_batch, 10 × 1 of A to an input control. relative 1.4.2 version Bowtie2 version 2.2.1 with parameters -p 4 -k 1. Peaks were called using MACS Picard tools. Raw sequencing data were mapped to the reference genome using Raw sequencers. data were to aligned the mm9 mouse reference genome using 10c Fig. Supplementary C a using studies, these in reported genes target overlapped motif) MYB a ing determined We contain results. peaks (MYB study our in identified ChIP-seq genes MYB-bound the MYB whether published other to mNSCs from ( H3K27ac to antibody with ment ( MYB to antibody Enrichment of MYB motifs was higher in significantly data generated with the tained a MYB motif ( that 92% of all peaks ( ( peaks across motifs all MYB to motifs and overexpressing MYB-QKI be identified the most enriched 10 × 1 of threshold detection (peak identified peaks the across 9 Fig. ( cells of these study a in previous genes target to be reported we performed MYB ChIP-seq in K562 cells and confirmed enrichment at genes 1 (Cell Signaling Technology, D5E4; optimized at 1 described 5 MYB of ChIP for used sheared chromatin among been (0.5 three ratios of antibody/chromatin previously has 45150 Abcam SAB4501936. Sigma and ab45150 Abcam MYB: for We antibodies two tested methodology ChIP-seqautomated our using ChIP-seq MYB for ratio 2 M M test with Yates correction. We observed significant enrichment ( enrichment significant We observed correction. Yates with test Supplementary Table 9 Supplementary ChIP-seq enrichment for H3K27ac marks was performed on human PLGGs ChIP libraries were indexed, pooled and sequenced on Illumina HiSeq 2000 Results from the MYB ChIP-seq analysis were validated in three ways. First, l of ab45150/1,000,000 cells to be optimal. be to cells ab45150/1,000,000 of l l of each antibody/1,000,000 cells) and performed ChIP-seq as previously previously as ChIP-seq performed and cells) antibody/1,000,000 each of l ) 6 2 . Second, we used HOMER used we Second, . −7 6 0 was used. The density of genomic regions was calculated using using calculated was regions genomic of density The used. was . . As a positive control, we included an antibody targeting H3K27ac P < 0.05 and and 0.05 < Cells were lysed and subjected to SDS-PAGE gradient gels as 5 9 P 3 was performed in GenePattern using default settings. settings. default using GenePattern in performed was 1 < 0.0001) as compared to those generated from enrich from generated those to compared as 0.0001) < Supplementary Fig. 10b Supplementary . Blots were probed with antibodies against MYB (ab45150, P 3 1 10 × 1 = threshold -value P $ . Primers for P q ) < 0.0001) ( 0.0001) < 64 < 0.35 were considered to have significant changes changes to have significant considered were < 0.35 < 0.05 were considered to be significantly altered. altered. significantly be to considered were 0.05 < $ . Samples were amplified in triplicate, and the data and the in triplicate, were . amplified Samples , C 6 5 t . method. 6 6 were downloaded and visualized in IGV. in visualized and downloaded were P = 1 6 2 3 4 Supplementary Fig. 10a Fig. Supplementary MYB to perform an unbiased motif analysis analysis motif unbiased an perform to . . Analysis was performed as above using −25 RNA with the was RNeasy extracted , ). Third, we compared our results results our compared we Third, ). P QKI M -value threshold for enrichment for enrichment threshold -value and and g of RNA using High-Capacity High-Capacity RNA using of g We systematically determined and and M −7 l/1,000,000 l/1,000,000 cells). We found Supplementary TableSupplementary 10 ; 3,392/3,672 peaks) con peaks) 3,392/3,672 ; 5 ATCB 8 . Comparative marker marker Comparative . doi: ( B Supplementary Supplementary 10.1038/ng.3500 2 -actin) -actin) are listed 3 ). We observed We). observed . We split the the split We . −7 ) in mNSCs mNSCs in ) M P l, l, 1 < 0.0001; M RNA l l and 60 , 6 1 ). ). ------.

© 2016 Nature America, Inc. All rights reserved. depict s.e.m. depict for performed was mouse analysis studies, and Kaplan-Meier curves were survival generated. The error bars shown (Mantel-Cox) Log-rank groups. tiple mul of comparison for used was correction ANOVAappropriate. with as P analysis. Statistical allocation. group to blinded were Neuropathologists measured. outcome primary the was tumorigenicity mouse identity. of assessment Sample size Qualitative was not predetermined. were used. mice 44 of total A used. Taconic were from mice IcrTac:ICRPrkdc-Scid male Four- to six-week-old by cells a of neuropathologist. atypical of identification basis the on present as scored were Tumors analysis. histological routine to were subjected Brains symptoms. of at onset neurological the and euthanized right striatum of immunocompromised ICR-SCID mice. Mice were monitored cells/ viable 100,000 at resuspended formula the using width × calculated 1/2(length = volume was volume tumor Ellipsoid biweekly. ured meas was Tumorgrowth mice. female and with male age, representationof of equal weeks 6–10 were Mice line). cell each for mice (five mice NSG lines. vivo In described previously as performed a doi: 47. 46. 45. 44. 43. values were calculated using Fisher’s exact tests, P Mouse injections were not randomized nor were experimenters blinded to blinded nor were experimenters were not randomized Mouse injections and dissociated were neurospheres injections, mouse intracranial For 10 × 1 of enrichment for threshold -value

Cibulskis, K. Cibulskis, Beroukhim, R. D.Y. Chiang, A. McKenna, Burrows-Wheeler with alignment read short accurate and Fast R. Durbin, & H. Li, heterogeneous cancer samples. cancer heterogeneous cancers. sequencing. parallel massively (2010). data. sequencing DNA next-generation analyzing transform. 10.1038/ng.3500 NIH3T3 cell lines were injected subcutaneously into the flanks of of flanks the into subcutaneously injected were lines cell NIH3T3 experiments. Nature Bioinformatics et al. et t al. et t al. et et al.

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