Control

IDH1 R

132H

mutan

t

OFF150

1000

2000

3000

4000

2HG

/D5-

2HG

*100

0

2HGb c

+dox

-dox

off

1P 5P 10P 20P 40P

Tet-inducible IDH1 R132H

off

-

+

-

Passages following Dox withdrawal at

baseline Baseline (P30)

+

d

Immortalized human astrocytes IDH1 R132H Tumorspheres

Mod

el

DOX inducible Constitutive TS603 (IDH1

R132H, 1p/19q) TS543, TS667

ChI

P-s

eq

H3K4me3, H3K36me3, H3K9me3, H3K27me3

H3K4me3, H3K36me3, H3K9me3,

H3K27me3, H4K20me3

H3K4me3

DN

A m

ethy

latio

n

(Illumina 450K) lllumina 450K lllumina 450K (TS603)

Gen

e ex

pres

sion

RNA-seq, Affymetrix Affymetrix RNA-seq

a

e

off d

ox 2

0P

off d

ox 4

0P

-dox

40P

-dox

1P

-dox

5P

-dox

10P

-dox

20P

off d

ox 1

0P

+dox

20P

+dox

40P

+dox

10P

off d

ox 5

P

+dox

1P

off d

ox 1

P

+dox

5P

h

4000

3000

2000

1000

0 2HG

/ D

5-2H

G ra

tio

Dox: - + off - + off - + off EV WT MUT

IDH1 R132H

IDH1 R132H

Figure 1

f

-dox

+dox

1P

5P

10P

20P

40P

-2 0 2

+dox

-dox

1P

5P

10P

20P

40P

Tran

sien

t G

radu

al

Per

s.

Tran

sien

t G

radu

al

Per

s.

10

9

8

7

6

mutIDHCD24

-

mutIDHCD24

+0

200

400

600

800

Num

ber o

f col

onie

s

*

Num

ber o

f col

onie

s

800

600

400

200

0

IDH1 R132H CD24-

IDH1 R132H CD24+

i j

2.5

5.0

7.5

8 pc

w

9 pc

w

12 p

cw

13 p

cw

16 p

cw

17 p

cw

19 p

cw

21 p

cw

24 p

cw

25 p

cw

26 p

cw

35 p

cw

37 p

cw

4 m

os

10 m

os

1 yr

s 2

yrs

3 yr

s 4

yrs

8 yr

s 11

yrs

13

yrs

15

yrs

18

yrs

19

yrs

21

yrs

23

yrs

30

yrs

36

yrs

37

yrs

40

yrs

Log2

(RP

KM

+ 1

)

NFE2L3 GNAL SCN3A INHBB CD24P4 L1CAM

LYN

Prenatal age Postnatal age

g

min

usplu

soff1 off5

off10

off20

off40

2

4

6

8

10

Exp

ress

ion

Persistent upregulated

L1CAMKRT15WNT2BMTUS1TMEM156WNT6WNT6

TMEM156 MTUS1

KRT15 L1CAM G

ene

Exp

ress

ion

(up-

regu

late

d)

WNT2B

10

8

6

4

2

min

usplu

soff1 off5

off10

off20

off40

2

4

6

8

10

Exp

ress

ion

Persistent downregulated

MEOX2MAFNKX2-1

min

usplu

soff1 off5

off10

off20

off40

2

4

6

8

10

Exp

ress

ion

Persistent downregulated

MEOX2MAFNKX2-1NKX2-1

MEOX2

Gen

e E

xpre

ssio

n (d

own-

regu

late

d)

MAF

10

8

6

4

2

k

H3K27

me3

H3K36

me3

H3K79

me2

H3K4m

e3

H3K9m

e3

H4K20

me30

20000

40000

60000

Parental stable linesN

umbe

r of P

eaks

21040

H3K79

me2

H3K36

me3

H3K4m

e3

H3K27

me3

H3K9m

e3

H4K20

me30

5

10

15

% G

enom

e C

over

age

IDH1 R132H stable lines

21040

5'UTR/TSSexonintron3'UTR/TTSIntergenicnon-coding

PAR02

PAR10

PAR40

MUT02

MUT10

MUT400

50

100

Enric

hmen

t of

H3K

4me3

pea

ks (%

)

P2 P10 P40

IDH1 R132H Parental

H3K

9me3

H3K

36m

e3

P2 P10 P40

a

b d

c

e f

IDH1 R132H %

Gen

ome

Cov

erag

e 15

10

5

0

IDH1 R132H

Num

ber o

f Pea

ks

H3K27

me3

H3K36

me3

H3K79

me2

H3K4m

e3

H3K9m

e3

H4K20

me30

50000

100000

150000

Num

ber o

f Pea

ks

IDH1 R132H stable lines

21040

150000

100000

50000

0

H3K27

me3

H3K36

me3

H3K79

me2

H3K4m

e3

H3K9m

e3

H4K20

me30

50000

100000

150000

Num

ber o

f Pea

ks

IDH1 R132H stable lines

21040Passage 40

Passage 10

Passage 2

Enr

ichm

ent o

f H

3K4m

e3 p

eaks

(%)

100

50

0 P2 P10 P40 P2 P10 P40

Parental IDH1 R132H

non-coding

intergenic

3’ UTR / TTS

intron

exon

5’ UTR/ TSS

H3K79

me2

H3K36

me3

H3K4m

e3

H3K27

me3

H3K9m

e3

H4K20

me30

2

4

6

8

10

% G

enom

e C

over

age

Parental stable lines

21040

10

8

6

4

2

0

% G

enom

e C

over

age

Parental

Num

ber o

f Pea

ks

60000

40000

20000

0

0

Parental

H3K27me3

H3K36me3

H3K79me2

H3K4me3

H3K9me3

H4K20me3

0

20000

40000

60000

Parental stable lines

Num

ber of Peaks

21040

Passage 2 Passage 10

Passage 40

P2

P10

P40 P2

P10

P40

Par

enta

l ID

H1

R

132H

P2 P10 P40 P2 P10 P40

Par

enta

l ID

H1

R

132H

DANCR

PDGFRA

H3K27me3 H3K36me3 H3K4me3 H3K9me30

50000

100000

150000

200000

Num

ber o

f Pea

ks

M30-M30+M70off

Num

ber o

f Pea

ks

200000

150000

100000

50000

0 H3K27me3 H3K36me3 H3K4me3 H3K9me3

0

50000

100000

150000

200000

Num

ber o

f Pea

ks

M30-M30+M70off

-dox

+dox off dox 40P

g Mutant Passage 2 Mutant Passage 10 Mutant Passage 40

H3K

36m

e3

H3K

9me3

H3K

27m

e3

H3K

4me3

Gen

ome

%

H3K

27m

e3

H3K

9me3

H3K

36m

e3

H3K

4me3

Gen

ome

%

H3K

27m

e3

H3K

36m

e3

H3K

9me3

H3K

4me3

Gen

ome

%

h

H3K

4me3

H3K

27m

e3

H3K

36m

e3

H3K

9me3

Gen

ome

%

H3K

27m

e3

H3K

9me3

H3K

36m

e3

H3K

4me3

Gen

ome

%

H3K

27m

e3

H3K

9me3

H3K

36m

e3

H3K

4me3

Gen

ome

%

-dox +dox off dox 40P

Figure 2

a b c

d

early

-inte

rmed

iate

gradual

persis

tent

0

5000

10000

15000

Num

ber

of L

oci

HypomethylatedHypermethylated

J

-dox

+d

ox

-dox

+dox

hyper

off dox 1P 0 2 4 6

0 2 4 6

0 2 4 6

0 2 4 6

0 2 4 6

0 2 4 6

-0.2

0.2

-0.4

0.4

Delta Beta

dens

ity

hypo

off dox 5P

off dox 10P

off dox 20P

off dox 40P

off d

ox 2

0P

off d

ox 4

0P

-dox

40P

-dox

1P

-dox

5P

-dox

10P

-d

ox 2

0P

off d

ox 1

0P

+dox

20P

+d

ox 4

0P

+dox

5P

+dox

10P

of

f dox

1P

+dox

1P

off d

ox 5

P

20

40

60

early

-inte

rmed

iate

gradual

persis

tent

0

5000

10000

15000

Num

ber

of L

oci

HypomethylatedHypermethylated

Num

ber o

f Loc

i

transient gradual persistent

15000

10000

5000

0

Hypomethylated

Hypermethylated

Transient

Gradual

Persistent

0.1

1

10

100 Low

Medium

High

Transient

Gradual

Persistent

0.1

1

10

100100

10

1

0.1 transient gradual persistent

Low

Medium High

100

10

1

0.1 transient gradual persistent

Per

cent

Tot

al

Per

cent

Tot

al

f

H3K4me3 H3K27me3 H3K36me3 H3K9me3 RNA-Seq

4 2 0 -2 -4

Methylation clusters ChIP-seq hype

r hy

po

Dow

nreg

ulat

ed

hypomethylated hypermethylated e

TSS *

* * * * * * * * * * *

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

10

20

30

40

50hypomethylated hypermethylated

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

50

10

30

40

20

Upe

gula

ted

1 – Active TSS 2 – Flanking active TSS 3 – Transcription at 5’ and 3’ 4 – Strong transcription 5 - Weak transcription

6- Genic enhancers 7 – Enhancers 8 – ZNF genes + repeats 9 – Heterochromatin 10 – Bivalent/poised TSS

11 – Flanking bivalent TSS/Enh 12 – Bivalent enhancer 13 – Repressed Polycomb 14 – Weak repressed Polycomb 15 – Quiescent / low

1P

5P

10P

20P

40P

1P

5P

10P

20P

40P

-2 0 2

Figure 3

Tran

sien

t G

radu

al

Per

sist

ent

Tran

sien

t G

radu

al

Per

sist

ent

g

minus_E1

minus_E2

minus_E3

minus_E4

minus_E5

minus_E6

minus_E7

minus_E8

minus_E9

minus_E10

0

50

100%

Tot

alIDH1 R132H Chromatin State Transitions

E1E2E3E4E5E6E7E8E9E10

Total=11443

3' UTR/TTS5' UTR/TSSintergenicexonintronnon-coding

<-500

[-500

,-50]

[-50,-

5][-5

,0] [0,5]

[5-50

]

[50-50

0]>5

000

1000

2000

3000

4000

Differentially enriched H3K4me3 peaksplus vs minus

Distance to TSS (kb)

Num

ber o

f Pea

ks

+dox off dox 40P -dox

-1

0

1

2

log 2(

Fold

cha

nge

vs in

put)

H3K4me3 Differential PeaksAverage Profile

MINUS M30PLUS M30OFF M70

-1000 -500 5' End 3' End 500 1000

Genomic Region (5'-> 3')

a b c

d e f

g

-dox

+dox

H3K

4me3

RN

A-seq

Chromatin State Transitions P

erce

nt T

otal

100

50

0 1 2 3 4 5 6 7 8 9 10 minus_

E1

minus_E2

minus_E3

minus_E4

minus_E5

minus_E6

minus_E7

minus_E8

minus_E9

minus_E10

0

50

100

% T

otal

IDH1 R132H Chromatin State Transitions

E1E2E3E4E5E6E7E8E9E10

1 2 3 4 5 6 7 8 9

10

+dox states

-dox states

MAP1LC3A

off dox 40P

-dox

+dox

off dox 40P

-4 4 0

-log10 FDR Q-value

0 10 30

H3K4me3 Differential Peaks Average Profile

Log 2

(Fol

d C

hang

e vs

Inpu

t)

2

1

0

-1

2

-1000 -500 5’ End 3’ End 500 1000 Genomic Region (5’ ->3’)

off dox 40P +dox -dox

Differentially enriched H3K4me3 peaks +dox vs -dox

4000

3000

2000

1000

0

Num

ber o

f Pea

ks

Distance to TSS (kb)

Total=11,443

3’ UTR / TTS 5’ UTR / TSS intergenic exon intron non-coding

h 5 0 5

Figure 4

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

NES=2.39 FDR q-val < 0.0001

0 5 10 15 20

0

5

10

15

20

ERV expression

Par40 -doxLog2(FPKM+1)

Par

40 +

dox

Log 2(

FPK

M+1

)

0 5 10 15 20

0

5

10

15

20

M40 -doxLog2(FPKM+1)

M40

+do

xLo

g 2(FP

KM

+1)

ERV expression

ERVK3-7

ERVW-3

ERVH-2

ERVH-4

ERVH-5ERVH-6

Enr

ichm

ent S

core

+d

ox v

s -d

ox

0.1 0.0

0.4 0.5

0.3 0.2

Enr

ichm

ent S

core

of

f dox

vs

-dox

NES=1.92 FDR q-val < 0.0001

Interferon Alpha Response

Interferon Alpha Response

chr1

chr2

chr3

chr4

chr5

chr6

chr7

chr8

chr9

chr1

0

chr1

1

chr1

2

chr1

3

chr1

4

chr2

1

chr1

9

chr1

7

chr1

5

chr1

6

chr1

8

chr2

0

chr2

2

0.4

0.2

0

-0.2

-0.4

-0.6

NES = -1.7362279 FDR q-val = 0.13

-0.1

-0.4

-0.3

-0.2

0.0

Chromosome 19q13

dox+ vs dox-

a

b

c d

e g

h i

+dox

40P

Lo

g 2 (F

PK

M+1

)

-dox IDH1 R132H 40P Log2 (FPKM+1)

ERV expression

-dox empty vector 40P Log2 (FPKM+1)

+dox

em

pty

vect

or 4

0P

Log 2

(FP

KM

+1)

ERV expression

20

15

10

5

0

0 5 10 15 20

20

15

10

5

0

0 5 10 15 20

0 1 2 3 4 5

M31_MINUSM31_OFF

M31_PLUSM70_MINUS

M70_OFFM70_PLUS

P31_MINUSP31_OFF

P31_PLUSP70_MINUS

P70_OFFP70_PLUS

TS_543TS_603TS_667

geo mean of viral defense signature

Geometric mean of FPKM

f TS667 TS603

+dox 40P off dox 40P

-dox 40P +dox

off dox 1P -dox

+dox 40P off dox 40P

-dox 40P +dox

off dox 1P -dox

TS543

Em

pty

Vect

or

IDH

1 R

132H

0 1 2 3 4 5

Geometric mean of FPKM

TS66

7 E

V -d

ox 4

0P

MU

T +d

ox 4

0P

TS60

3 M

UT

-dox

40P

EV

off

dox

40P

EV

+do

x 40

P

MU

T –d

ox

MU

T of

f dox

40P

TS

543

MU

T of

f dox

1P

MU

T +d

ox

EV

-dox

E

V +

dox

EV

off

dox

1P

-3 3

IDH1 R132H +dox vs -dox

% M

etap

hase

s w

ith

rela

tive

loss

of 1

9q

-dox+d

ox-d

ox+d

ox0

10

20

30

40

% M

etap

hase

s w

ith

rela

tive

loss

of 1

9q

P30P35

40

30

20

10

0

Figure 5

1 Passage off dox

0 50 100 1500

2

4

6

8

Time (hours)

Cel

l ind

ex

6 Passages off dox

0 50 100 1500

2

4

6

8

Time (hours)

Cel

l ind

ex

12 passages off dox

0 50 100 1500

2

4

6

8

10

Time (hours)

Cel

l ind

ex

whole brain surface

a b

c

3D tumor reconstruction

Z =1.245 mm Z = 0.945 mm Z = 1.345 mm

Cel

l Ind

ex

Time (hours)

Cel

l Ind

ex

Time (hours) 0 50 100 150

0

2

4

6

8

0

2

4

6

8

50 100 150 0

0

2

4

6

8

10

0 50 100 150

16 Passages off dox

0 50 100 1500

2

4

6

8

Time (hours)

Cel

l ind

ex

0 50 100 150 0

2

4

6

8

1P off dox 5P off dox

12P off dox 15P off dox

-dox +dox off dox Av

erag

e R

adia

nce

(p

/s/c

m2 /s

r)

Time (hours) Time (hours)

Time (days)

0 13 27 35 44 56 69 83 99 128

103

104

105

106

107

Time (days)

Average'Ra

diance'(p

/s/cm2/sr)

-dox+doxoff dox

107

106

105

104

103

0 13 27 35 44 56 69 83 99 128

-dox +dox off dox

Cortex Striatum Hypothalamus

Per

cent

sig

nal

per a

rea

Per

cent

sig

nal

per a

rea

Per

cent

sig

nal

per a

rea

+dox off dox

MO SS ILA CP ACB FS OT AAA PALv LHA LPO

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

-dox +dox off dox

-dox

+dox

off dox

d

e

Figure 6