c d tel chromosome 15q11-q13 mkrn3 gel2 ndn ap1 -snrpn snornas/ ipw ube3a 0a abrb3 abra5g3 a2 c2 bp1...

1 2 3 4 5

6 7 8 11 129 10

13 14 15 17 1816

19 20 21 X22 Y

0

1,000

2,000

3,000

4,000

5,000

6,000

POU5F1

SOX2

DNMT3

B

NANOGLI

N28

TDGF1

WT-ESCsWT-iPSCsPWS-iPSCs

Rel

ativ

e ex

pres

sion

0

5

10

15

20

170

DES

NCAM1

AFP

PWS-iPSCsPWS-20 d EBs

Rel

ativ

e ex

pres

sion

a

1 2 3 4 5

6 7 8 11 129 10

13 14 15 17 1816

19 20 21 X22

PWS-iPSC-1 PWS-iPSC-2

PWS-iPSC-1

PWS-iPSC-2

b

c d

Rel

ativ

e ex

pres

sion

0

25

50

75

100

WT-F

ib

PWS-F

ib0

100

200

300

400

500

WT-F

ib

PWS-F

ib0

500

1,000

1,500

2,000

WT-F

ib

PWS-F

ib

CRHBP(chromosome 5)

ZNF280D (chromosome 15)

RPPH1 (chromosome 14)

e

Supplementary Figure 1. Characterization of PWS-iPSCs. (a) G-band karyotyping of representative PWS-iPSC lines, demonstrating a diploid genome, excluding chromosome 15 aberrations. (b) Representative alkaline phosphatase staining for PWS-iPSC lines. Scale bar, 200 µm. (c) Mean expression levels ± s.d. of representative pluripotency markers in four PWS-iPSC lines (PWS-iPSC-1-A and B and PWS-iPSC-2-A and B) compared with �ve control (WT) PSCs (two iPSC and three ESC lines). (d) Gene expression analysis of 20-d-old embryoid bodies (EBs) from the two PWS-iPSC lines, demonstrating differentiation in vitro into all three embryonic germ layers. DES, NCAM1 and AFP represent markers for mesoderm, ectoderm and endoderm, respectively (mean expression levels ± s.d.; two biological replicates). (e) Mean expression levels ± s.d. of representative potential targets of PEGs in the PWS locus, in PWS and control (WT) �broblasts.

Nature Genetics: doi:10.1038/ng.2968

Cen Tel

Chromosome 15q11-q13

MKRN3

MAGEL2

NDNNPA

P1

SNURF-SNRPN

snoR

NAs/IPW

UBE3A

ATP10

A

GABRB3

GABRA5

GABRG3

OCA2

HERC2

ICBP2 BP3BP1

1 Mb

PAR5

Maternally expressedPaternally expressed

Case 3 PWS-iPSC-3 (maternal UPD 15) Paternal chromosome 15

a

b

0

−2

−4

−6

q11.

2q1

2q1

3.1

q14

q15.

1q2

1.1

q21.

2q2

1.3

q23

q24.

1q2

5.1

q26.

2q2

6.3

q13.

3q2

2.2

q22.

31

q25.

2q2

5.3

q26.

1

Chromosome 15q

log 2

exp

ress

ion

ratio

(PW

S-iP

SC

-3 /

WT

-PS

Cs)

q12

q11.

2q1

3.1

q21.

1q2

1.2q2

1.3q2

2.1

q22.

3q2

3.1q2

3.2q2

3.3q2

4.1q2

4.2

q24.

3q3

1.1

q31.

3

q32.

11

q32.

12

q32.

13q3

2.2

q32.

32

q32.

33

02468

1012

Exp

ress

ion

ratio

(PW

S-iP

SC

-3 /

WT

-PS

Cs)

Chromosome 14q

c

Supplementary Figure 2. Genome-wide gene expression analysis in PWS-iPSCs derived from a patient harboring a maternal UPD of chromosome 15. (a) Establishment of PWS-iPSCs with maternal UPD in the PWS region. (b,c) Analysis of gene expression by moving average plot along chromosome 15q (b, logarithmic scale) and 14q (c) in PWS-iPSC-3 relative to normal (WT) PSCs. Vertical dashed lines mark the genomic regions of the PWS (b) and DLK1-DIO3 (c) loci.


RTL1

Rel

ativ

e ex

pres

sion

Contro

l0

0.2

0.4

0.6

0.8

1.0

1.2

+siR

NA0

0.2

0.4

0.6

0.8

1

1.2MIR370

Contro

l

+siR

NA0

0.2

0.4

0.6

0.8

1

1.2MEG3

Contro

l

+siR

NA0

0.2

0.4

0.6

0.8

1

1.2MIR409

Contro

l

+siR

NA

a

−5

−4

−3

−2

−1

0

log 2

RTL

1 ex

pres

sion

rat

io(P

g-iP

SC

s / W

T-P

SC

s)b

miRNA-371-3(chromosome 19)

0

1

2

3

4

5

WT-P

SCs

Pg-iP

SCs

PWS-iP

SC-1

PWS-iP

SC-2

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

WT

-PS

Cs

Pg-

iPS

Cs

PW

S-iP

SC

s

PLAGL1 (chromosome 6)

0

50

100

150

200

250PEG10 (chromosome 7)

0200400600800

1,0001,2001,4001,600

WT

-PS

Cs

Pg-

iPS

Cs

PW

S-iP

SC

s

IGF2 (chromosome 11)

020406080

100120140160

WT

-PS

Cs

Pg-

iPS

Cs

PW

S-iP

SC

s

c d

Supplementary Figure 3. Analysis of known imprinted genes in PWS-iPSCs. (a) RTL1 expression ratio (in RPKM, logarithmic scale, two biological replicates), demonstrating its marked downregulation in Pg-iPSCs relative to normal PSCs. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) of RTL1, MEG3, miR-370 and miR-409 in normal PSCs before and after knockdown of RTL1 (+siRNA). (c) qRT-PCR of the mean relative fold change ± s.d. of miR-371-3 in normal PSCs, Pg-iPSCs and PWS-iPSCs (two biological replicates each), demonstrating comparable expression levels of miR-371-3 in PWS-iPSCs and normal PSCs. (d) Mean expression levels ± s.d. of representative known PEGs in distinct loci. Analyses were conducted on three PWS-iPSC lines (PWS-iPSC-1-A and B and PWS-iPSC-2-A), three Pg-iPSC lines (Pg-iPSC-A-11, 20 and 26) and �ve control PSCs (two iPSC and three ESC lines). WT-PSCs, normal PSCs.


Rel

ativ

e M

EG

3 ex

pres

sion

0

0.2

0.4

0.6

0.8

1.0

1.2

PWS fib

robla

sts

PWS B

-cell

line

1

PWS B

-cell

line

2

N.E. N.E.

Exp

ress

ion

ratio

(PW

S-F

ib/W

T-F

ib)

q12

q11.

2q1

3.1

q21.

1q2

1.2

q21.

3q2

2.1

q22.

3q2

3.1

q23.

2q2

3.3q2

4.1q2

4.2

q24.

3q3

1.1

q31.

3

q32.

11

q32.

12

q32.

13q3

2.2

q32.

32

q32.

33

0123456

Chromosome 14q

0

200

400

600

800

1,000

1,200

Rel

ativ

e ex

pres

sion

Fibroblasts iPSCsWTPgPWS

MEG3

SNORD113-

3

SNORD113-

4

SNORD114-

2

SNORD114-

3

SNORD114-

26

MEG3

SNORD113-

3

SNORD113-

4

SNORD114-

2

SNORD114-

3

SNORD114-

26

a

c

b

Supplementary Figure 4. Gene expression analyses in PWS cell lines. (a) Analysis of gene expression by moving average plot along chromosome 14q in PWS parental �broblasts relative to control �broblasts. Vertical dashed lines mark the genomic boundaries of the DLK1-DIO3 region. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in MEG3 in mature PWS cells derived from three affected individuals, showing negligible expression levels in patient-derived B cells. N.E., not expressed. (c) Expression analysis of all MEGs represented in the expression arrays in the parental �broblasts (left panel) and undifferentiated iPSCs (right panel). WT, control.


gDNA

cDNA

MEG3


TA C ATG CT

TA C ATG T

TA C ATG CT

TA C ATG T T T G GCG T

CTT T G GCG

T T G GCG T

T T G GCG CT


RTL1

Supplementary Figure 5. Proper monoallelic expression of imprinted genes in the DLK1-DIO3 locus in PWS-iPSCs. Direct sequencing of the transcribed region of MEG3 and RTL1 in genomic DNA (gDNA) and complementary DNA (cDNA) in PWS-iPSCs derived from three patients. Note that both MEG3 and RTL1 are expressed in a monoallelic fashion.


0

200

400

600

800

1,000

1,200

PWS-iP

SCs

PWS-iP

SCs

+ HOTA

IR

Rel

ativ

e H

OTA

IR e

xpre

ssio

n

0

0.5

1.0

1.5

Rel

ativ

e M

EG

3 ex

pres

sion

Con

trol

+ H

OTA

IR

+ IP

W

WT

-PS

Cs

PWS-iPSCs

0

50

100

150

WT

-iPS

Cs

Con

trol

+ IP

W

Con

trol

+ IP

WPWS-iPSCs

Pg-iPSCs

Rel

ativ

e Z

FP

57 e

xpre

ssio

nInput

H3K9me3

WT-ESCs

T CGAGT TG

T CGAGT TG

a b

c d

Supplementary Figure 6. Chromatin regulation in PWS-iPSCs. (a) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in HOTAIR in PWS-iPSCs before and after transfection with an overexpression plasmid. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in MEG3 in PWS-iPSCs before and after transfection with the HOTAIR overexpression plasmid, relative to control (WT) PSCs. (c) Allelic sequence analysis following ChIP for H3K9me3 reveals the association of this histone modi�cation with both methylated and unmethylated alleles of the IG-DMR in normal PSCs. (d) qRT-PCR of the mean relative fold change ± s.d. in ZFP57 in Pg-iPSCs (three biological replicates each) and PWS-iPSCs (three biological replicates each) following introduction of IPW, relative to control (WT) iPSCs.


Supplementary Table 1. Genes downregulated at least 3-fold in both PWS-iPSCs

and Pg-iPSCs compared with WT-iPSCs

Probe Set ID Gene Symbol Chromosome Start Stop WT-ESCs WT-iPSCs Pg-iPSCs PWS-iPSCs

7981990 SNORD116-21 15 25333951 25334042 3357.58 2621.19 78.77 9.28

7981976 SNORD116-14 15 25325289 25325380 3712.52 3214.09 90.59 17.33

7981982 SNORD116-17 15 25328735 25328826 5370.81 4760.38 162.51 33.68

7981986 SNORD116-17 15 25331674 25331765 5370.81 4760.38 162.51 33.68

7981943 PAR5 15 25230247 25230313 938.75 1057.28 32.62 10.14

7981964 SNORD116-8 15 25315578 25315673 1221.37 1769.01 32.19 17.53

7981958 SNORD116-5 15 25307480 25307574 1099.29 1294.71 22.93 13.61

7981962 SNORD116-5 15 25312935 25313029 1099.29 1294.71 22.93 13.61

7981953 SNORD116-3 15 25302007 25302101 1627.13 2351.93 48.93 24.86

7981966 SNORD116-3 15 25318254 25318348 1627.13 2351.93 48.93 24.86

7981978 SNORD116-15 15 25326434 25326525 6026.03 5255.39 177.84 57.66

7981988 SNORD116-20 15 25332809 25332900 2472.09 2160.71 67.20 24.79

7981996 SNORD116-24 15 25339184 25339275 700.86 587.50 16.64 8.00

7981955 SNORD116-4 15 25304685 25357041 675.72 790.81 27.75 13.34

7981949 SNORD116-1 15 25296624 25296718 501.63 585.67 14.30 10.92

7981919 SNRPN 15 25068794 25244175 1945.02 2189.04 90.59 50.38

7915592 --- 1 45196727 45196842 265.82 495.73 9.75 17.15

7981960 SNORD116-6 15 25310173 25310268 339.13 273.23 14.73 9.62

7981992 SNORD116-22 15 25335070 25335161 295.64 194.74 9.50 7.73

7981947 SNORD109A 15 25287120 25287187 207.84 221.88 14.00 12.12

7982098 SNORD109A 15 25523489 25523556 207.84 221.88 14.00 12.12

7981945 SNRPN 15 25281106 25281637 133.83 129.37 8.26 7.12

7982002 SNORD116-27 15 25346722 25346813 209.24 202.29 13.73 11.36

7924307 --- 1 215803368 215803459 144.20 118.68 11.02 7.65

7981984 SNORD116-18 15 25330532 25330623 172.99 103.74 7.94 6.85

7981974 SNORD116-13 15 25324205 25324296 107.31 92.77 7.69 6.60

7982006 SNORD116-29 15 25351668 25351750 150.15 128.34 14.30 11.81

7981951 SNORD116-2 15 25299357 25299451 223.14 180.75 37.15 25.29

7982000 SNORD116-26 15 25344646 25344741 51.04 58.20 9.83 8.57

7977507 RPPH1 14 20811233 20811565 1582.20 1712.27 382.87 257.26

7982004 SNORD116-28 15 25349789 25349878 129.21 74.16 15.81 12.07

7981994 SNORD116-23 15 25336932 25337024 154.04 60.77 12.03 10.14

7981970 SNORD116-11 15 25321076 25321167 74.04 62.66 13.50 10.49

8115443 --- 5 156085598 156087446 1976.07 1292.76 266.14 230.95

8133876 CD36 7 80231523 80306326 34.23 131.00 24.83 23.42

7978801 MDGA2 14 47309295 48143953 54.61 143.32 41.45 26.88

8094870 SHISA3 4 42399856 42403656 105.86 653.04 173.27 125.83

7981998 SNORD116-25 15 25342810 25342901 52.22 47.96 11.30 10.16

8124684 ZFP57 6 29640260 29658489 42.15 621.85 48.42 157.20

7986757 NDN 15 23930554 23932450 107.91 97.22 25.61 26.01

8095299 --- 4 60698893 60698992 26.54 62.04 17.00 17.21

8047784 ZDBF2 2 207139528 207172525 137.14 315.92 33.32 96.23

8134384 DYNC1I1 7 95401866 95727738 80.32 296.42 75.29 95.27


Supplementary Table 2. Genes upregulated at least 3-fold in both PWS-iPSCs and

Pg-iPSCs compared with WT-iPSCs

Probe Set ID Gene Symbol Chromosome Start Stop WT-ESCs WT-iPSCs Pg-iPSCs PWS-iPSCs

7976814 SNORD114-2 14 101418190 101418270 10.37 15.37 168.00 116.68

7976795 MEG3 14 101296942 101327358 71.51 81.31 628.81 636.23

7976810 SNORD113-3 14 101396247 101396327 27.02 18.70 313.84 172.13

7976812 SNORD113-4 14 101402828 101402902 8.62 19.13 513.70 203.52

7976816 SNORD114-3 14 101419686 101419760 15.61 19.48 138.20 550.09

7989159 ZNF280D 15 56922374 57030101 30.66 54.31 192.49 198.16

8124380 HIST1H1A 6 26017248 26018048 214.27 173.14 1531.35 654.25

8151931 TSPYL5 8 98285714 98290176 30.50 35.76 301.83 151.38

8027266 --- 19 20480241 20482121 585.71 488.66 1967.53 2218.60

8070629 C21orf105 21 44339133 44345726 228.59 135.80 462.47 822.77

8106418 CRHBP 5 76248680 76265299 96.88 63.27 245.57 1208.75

7976826 SNORD114-26 14 101453371 101453454 27.64 23.12 73.66 448.05


Supplementary Table 3. Complete list of primers

Quantitative RT-PCR

Gene 5′ primer

/ TaqMan probe 3′ primer

Size (bp)

Number of cycles

Tm (°C)

MEG3 GCCAGCTGTCCCTCTTACCTAA

GCAGGTGAACACAAGCAAAGAC

138 40 60

IPW TGCCTAGACCACCCACTAAAGG

AGTCTCCATGCGGAAGGAAGA

120 40 60

DES CTACACCTGCGAGATTGACG

TGTTGTCCTGGTAGCCACTG

110 40 60

NCAM1 GATGCGACCATCCACCTCAA

TCTCCGGAGGCTTCACAGGTA

113 40 60

AFP AGGCCAGGAACAGGAAGTCT

CAAAGCAGCACGAGTTTTTG

70 40 60

RTL1 TCCTGGCCATAAGAAAGCAC

ACCGAAGATGTGTGGAAAGC

158 40 60

miR-371-3 TGTCCAAGTGGAAAGTGCTG

AGGTTTTCCGGAGCCATTAC

177 40 60

GAPDH AGCCACATCGCTCAGACACC

GTACTCAGCGCCAGCATCG

302 40 60

ZFP57 TGTGGCCAGAATCTTTCTGCAT

AGCGCCTGCTGAAACATTTG

317 40 60

HOTAIR GGTAGAAAAAGCAACCACGAAGC

ACATAAACCTCTGTCTGTGAGTGCC

170 40 60

MIR370 Tm2275 40 60

MIR409 Tm2332 40 60

GAPDH Hs99999905_m1 40 60

PCR and RT-PCR for direct sequencing

Gene 5′ primer 3′ primer Size (bp)

Number of cycles

Tm (°C)

MEG3 TGAGGGGTTTTGAGTCATCC

GGAGTCAGGTGCCCTTATGA

510 35 60

RTL1 TGCAAGACCTCAAAGAGCAA

GCAGGATGAGCACAATCAGA

537 35 60

IG-DMR TCCTTGGACAAGAGAGCACA

GTTGCAAACCAGGGTGAAGT

197 35 60

IPW RT-PCR and cloning

Gene 5′ primer 3′ primer Size (bp)

Number of cycles

Tm (°C)

IPW

AGCTGCTAGCCCAAATCATAGATCAAGATATTTTATTTATGTATG

ACGTCCCGGGTTACACAGGTTTAAGAATTTATTAAGAAAAGA

4498 39 55

ChIP-qPCR

Region 5′ primer 3′ primer Size (bp)

Number of cycles

Tm (°C)

DLK1 promoter AGGCCCTGACTGAGATGCTG

AGACGCTTACGCAGAGCCA

121 40 60

IG-DMR ACTGGAACTGGCAAAGCACC

ACATCAAAAATGCGGAACTCG

121 40 60

SNORD-113 cluster AAAGAACTCACCCGGGCAAT

GCCTCACACCAGCTGAGAGAC

121 40 60

Negative control (chromosome 14)

TGGCCTGCATCCTGTTTTATC

AGACTGACACCTGCTGGCCT

121 40 60


c d tel chromosome 15q11-q13 mkrn3 gel2 ndn ap1 -snrpn snornas/ ipw ube3a 0a abrb3 abra5g3 a2 c2 bp1...

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