Cell Stem Cell

Supplemental Information

Erosion of X Chromosome Inactivation in Human

Pluripotent Cells Initiates with XACT Coating and

Depends on a Specific Heterochromatin Landscape

Céline Vallot, Jean-François Ouimette, Mélanie Makhlouf, Olivier Féraud, Julien Pontis,

Julien Côme, Cécile Martinat, Annelise Bennaceur-Griscelli, Marc Lalande, and Claire

Rougeulle

Supplemental*Information*

Erosion' of' X' chromosome' inactivation' in' human' pluripotent' cells' initiates'with'XACT'coating'and'depends'on'a'specific'heterochromatin'landscape*

!

Céline!Vallot1,2,!6,! Jean1François!Ouimette1,2,6,!Mélanie!Makhlouf1,!2,!Olivier!Féraud3,! ! Julien!Pontis1,2,!

Julien!Côme4,!Cécile!Martinat4,!Annelise!Bennaceur1Griscelli3,!Marc!Lalande5!and!Claire!Rougeulle*1,2!

*

Contents:*

I.*Supplemental*Figures*and*Tables*

II.*Supplemental*Experimental*Procedures*

III.*Supplemental*References*

*

! 2!

!I.*Supplemental*Figures*and*Tables*

400 10 20 30% occupied

XaYDuodenum Muscle

Figure S1: Epigenomic analyses (Related to Figure 1).(A) Zoom on the XIST neighboring region, with genomic coordinates indicated (in Mb, hg19) and genes represented as black boxes. H3K27me3 (green) and H3K9me3 (blue) ChIP-seq data in XaXi H9 cells is represented as log2 enrichment ratio of IP over input. (B, C) H3K27me3 and H3K9me3 occupancy of the active X chromosome and autosomes in one additional female (Colonic Mucosa cells) and four male differentiated cell types (AG04450 male lung cells, Duodenum Mucosa cells, Duodenum Muscle cells and Pancreas cells). ChIPseq were down-sampled to the smallest sample for each category.(D, E) Scatter plots comparing H3K27me3 (D) and H3K9me3 (E) log2 enrichment over input for 100kb bins on the X chromosome between XaXi pluripotent (H9) and differentiated cells (IMR90). (F, G) Euler diagrams displaying Mb of X chromosome covered by H3K27me3 peaks (F) or H3K9me3 peaks (G) in XaXi H9 cells versus IMR90 cells.

A

B

400 10 20 30% occupied

log2 IP/INPUT IMR90

log2

IP/IN

PUT

XaXi

H9

log2 IP/INPUTIMR90

E

C

3"

XaYLung

H3K9me3

H3K27me3

XaXiH9

XIST

(Mb)

73,2572,75

0

4

4H3K9me3

H3K27me3

JPXTSIXCHIC1

NAP2K4P1

CDX4

H3K27me3XaY

Duodenum Mucosa

H3K9me3

0 10 20 30 40% occupied

XaYDuodenum Mucosa

73

4

2

0

420-2-2

4

2

0

420-2

-2

r=0.35 p<10-6

r=0.44 p<10-6

0 10 20 30 40% occupied

chr22chrX

chr21

chr19chr20

chr18

chr16chr17chr15

chr13chr14

chr12

chr10chr11

chr9

chr7chr8

chr6

chr4chr5

chr3

chr1chr2

XaXiColonic Mucosa

XaXiColonic Mucosa

0 10 20 30 40% occupied

chr22chrX

chr21

chr19chr20

chr18

chr16chr17chr15

chr13chr14

chr12

chr10chr11

chr9

chr7chr8

chr6

chr4chr5

chr3

chr1chr2

D

0 10 20 30% occupied

40

XaYPancreas

400 10 20 30% occupied

XaYLung

400 10 20 30% occupied

400 10 20 30% occupied

XaYPancreas

400 10 20 30% occupied

XaYDuodenum Muscle

20.70

23.66

F

GH3K9me3

H3K27me3 lo

g2 IP

/INPU

T Xa

Xi H

9

Figure S2: H3K27me3 and H3K9me3 profiles in hESCs and differentiated cells (Related to Figure 1 and 2).(A-D) Superposition of H3K9me3 and H3K27me3 ChIP-seq for XaXi hESC (A), differentiated normal cells (B), immortalized cells (C) and XaXe WIBR3 hESC. Log2 ratios were compared using Pearson correlation test and the associated p-value was calculated using random permutations of the data sets.(E) Scatter plots of log2 enrichment over input for 100kb bins along the X chromosome for H3K27me3 and H3K9me3 comparing our IMR90 ChIPseq with datasets from the UCSD Human Reference Epigenome Mapping Project (GSE16256). For H3K27me3 ChIPseq (left panel), the same antibody was used in both experiments, and the two datasets display high correlation (r=0.87, p<10-6). For the H3K9me3 ChIPseq (right panel), we used a different antibody from the one previously used, which could explain the variations observed bewteen the two datasets.

IMR90 (Vallot, Ouimette et al.)Upstate 07-449

IMR

90 (R

en L

ab)

Ups

tate

07-

449

IMR90 (Vallot, Ouimette et al.)Diagenode pAb-056-050

IMR

90 (R

en L

ab)

Abc

am a

b889

8

4"

corr= −0.5

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 ra

tio RP

M IP/R

PM INP

UT

K9_HUES6_sample199_SRR1067001.100k.bedgraph K27_HUES6_sample199_SRR1067002.100k.bedgraph INP_HUES6_sample199_SRR1067000.100k.bedgraphr= - 0.56 p<10-6

log2

IP/in

put

0

2

4

-2

(Mb)50 100 1500

chrX

r= - 0.64 p<10-6

log2

IP/in

put

(Mb)

0

2

4

-250 100 1500

chrX

XaXi HUES6 XaXi HUES48corr= −0.6

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 rat

io RPM

IP/RP

M INP

UT

K9_HUES48_sample197_SRR1067095.100k.bedgraph K27_HUES48_sample197_SRR1067514.100k.bedgraph INP_HUES48_sample197_SRR1067093.100k.bedgraph

corr= 0.02

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 rat

io RPM

IP/RP

M INP

UT

GSM669968_BI.Adipose_Nuclei.H3K9me3.92.100k.bedgraph GSM669930_BI.Adipose_Nuclei.H3K27me3.92.100k.bedgraph GSM669934_BI.Adipose_Nuclei.Input.92.100k.bedgraph

corr= −0.0

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 ra

tio RP

M IP/R

PM INP

UT

GSM621668_BI.Colonic_Mucosa.H3K9me3.32.100k.bedgraph GSM621673_BI.Colonic_Mucosa.H3K27me3.32.100k.bedgraph GSM621669_BI.Colonic_Mucosa.Input.32.100k.bedgraph

log2

IP/in

put

0

2

4

-2

(Mb)50 100 1500

chrX

XaXi Adult adipocyte r= 0.02 p=0.12

log2

IP/in

put

r= - 0.10 p<10-6

(Mb)

0

2

4

-250 100 1500

chrX

XaXi Adult colonic mucosa

corr= −0.4

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 rat

io RPM

IP/RP

M INP

UT

GSM613875_UCSF−UBC.Breast_vHMEC.H3K9me3.RM035.HS2617.100k.bedgraph GSM669594_UCSF−UBC.Breast_vHMEC.H3K27me3.RM035.100k.bedgraph GSM613892_UCSF−UBC.Breast_vHMEC.Input.RM035.HS2620.100k.bedgraph

corr= −0.7

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 ra

tio RP

M IP/R

PM INP

UT

K9_RPEhTERT_DRR003585.100k.bedgraph K27_RPEhTERT_DRR003586.100k.bedgraph INP_RPEhTERT_DRR003583.100k.bedgraph

log2

IP/in

put

r= - 0.47 p<10-6

0

2

4

-2

(Mb)50 100 1500

chrX

XaXi vHMEC

log2

IP/in

put

r= - 0.74 p<10-6

(Mb)

0

2

4

-250 100 1500

chrX

XaXi RPE-hTERT

corr= 0.05

−2

0

2

4

0.0e+00 5.0e+07 1.0e+08 1.5e+08V3

log2 ra

tio RP

M IP/R

PM INP

UT

K9_WIBR3_SRR1035437.100k.bedgraph K27_WIBR3_SRR1035435.100k.bedgraph INP_SRR1035438.100k.bedgraph

log2

IP/in

put

r= 0.06 p=0.001

0

2

4

-2

(Mb)50 100 1500

chrX

XaXe WIBR3

A

B

H3K27me3H3K9me3

H3K27me3H3K9me3

C

D

H3K27me3H3K9me3

H3K27me3H3K9me3

H3K27me3H3K9me3

H3K27me3H3K9me3

H3K27me3H3K9me3

E

r=0.69 p<10-6

H3K9me3

20-2

2

0

-2r=0.87 p<10-6

H3K27me3

20-2

2

0

-2

A

lnc103

71%

XaXi H9 XaXe H9

90%

lnc103

p<10-8 p=0.002 p<10-6

XaXiXaXe

XaXiXaXe

XaXiXaXe

SNP1 SNP2 SNP3

% re

ads

0%

25%

50%

75%

100%

lnc121

76%

XaXi H9 XaXe H9

86%

lnc121

% re

ads

0%

25%

50%

75%

100%

SNP1 SNP2 SNP3 SNP4

p=0.009 p<10-4 p<10-3 p=0.002

Allele AAllele B

Allele AAllele B

Clnc103102,98 103 103,02

XaXe H9 RNAseq

(+ strand)

H1 RNAseq(+ strand)

K4me3 Raw Signal

K36me3 Raw Signal

(Mb)

** *SNP1

SNP2SNP3

121,36 121,38121,34 121,40 (Mb)

lnc121

* * *SNP1 *SNP2

SNP3SNP4

Figure S3: Validation of allelic RNA-seq (Related to Figure 3).(A) Assessment of allelic expression by pyrosequencing for TSPAN6, POLA1 and CTPS2. (B, C) Identification of two lncRNAs relaxed from XCI in XaXe cells, lnc103 and lnc121. Upper panel: we compared our RNA-seq data (XaXe H9) to published strand-specific RNA-seq from H1 hESC (UCSC accession number: wgEncodeEH000132). Both show transcription originating from the plus strand over two large intergenic regions. ChIP-seq data for H1 hESC from the ENCODE project shows large H3K36me3 peaks, usually found within the body of transcribed genes, over the identified transcription unit. Furthermore, one large H3K4me3 peak is located at the 5' end of each region. We used the H3K36me3 peak calling to delineate the two lncRNA genes: lnc103 (102,995,140-103,055,964) and lnc121 (121,352,438-121,385,472). Middle panel: Allelic counts assessed by RNA-seq in XaXi and XaXe H9 cells were compared using a Fisher’s exact test (corresponding p-values are indicated above each SNP). Lower panel: reactivation observed by RNA-seq was confirmed by RNA-FISH using fosmids spanning each lncRNAs in XaXi and XaXe H9 cells. Predominant expression pattern is displayed for XaXi and XaXe H9 cells, numbers of nuclei with mono or biallelic pattern were compared using a Fisher’s exact test. White scale bars represent 5 µm.

XaXiXaXe

XaXiXaXe

XaXiXaXe

XaXiXaXe

B

0%

20%

40%

60%

80%

100%

gDNA XaXi H9 XaXe H9

TSPAN6 monoallelic

CTPS2escapee

0%

20%

40%

60%

80%

100%

gDNA XaXi H9

XaXe H9

Allele BAllele A

POLA1reactivated

0%

20%

40%

60%

80%

100%

gDNA XaXi H9

XaXe H9

5"

0

5

0

100

0

15

0

10

0

10

0

30

0

30

0

0,25

Figure S4: X chromosome paint RNA-FISH to monitor XCI erosion (Related to Figure 4). (A) 5µl of X-paint (yellow) were co-precipitated with various amounts of human Cot-1 DNA prior to over-night hybridization (see Experimental Procedures). White scale bars represent 5µm.(B) X-paint (yellow) in adult female fibroblasts (AG09603). White scale bars represent 5µm.(C) ChIP-seq data of the ATRX locus for H3K27me3 and H3K9me3 in XaXi and XaXe H9 cells. The charts shows the log2 enrichment signal of IP over input over 10kb binning windows. (D) Allelic expression analyzed by RNA-FISH using a BAC probe covering ATRX (green) in XaXi and XaXe H9 cells. White scale bars represent 5µm. The barchart displays the percentage of nuclei with mono and bi-allelic pattern of expression (n=100). (E) Validation of RNA FISH chromosome paint approach using an independent X-paint (yellow; Cambio). (F) X-paint (yellow) in XaXe HUES1. (G) X-paint RNA-FISH (yellow) followed by X-paint DNA-FISH (green) in XaXe H9 cells. White scale bars represent 5µm.

1µg/µl Cot-10µg Cot-1 2µg/µl Cot-1

fibro

blas

t

X-paint RNAA B

X-paint RNA #2

XaXe

Xa

Xe

X-paint DNAX-paint RNA

Xa

Xe

E G

XaXi

H9

XaXe

H9

XaXe

H9

F

XaXe

HU

ES1

81%Xa

XeX-paint RNA

X-paint RNA X-paint RNA X-paint RNA

6"

100%

% o

f nuc

lei

0%

20%

40%

60%

80%

XaXi XaXe

XaXe H9XaXi H9

bi-allelicmono-allelic

XaXi H9XaXe H9

XaXi H9XaXe H9

Mb76 7775chrX

ATRX MAGT1LOC101928469

0

4

4log2

IP/in

put

100%

C

D

100%ATRX100%ATRX

H3K27me3

H3K9me3

Figure S5: Additional monitoring of XCI erosion during differentiation (Related to Figure 5).(A) Characterization of NSCs derived from XaXe HUES1 by immunofluorescence using NESTIN (green) and SOX2 (red) antibodies. The numbers in white indicate the percentage of positive cells as quantified using ArrayScan. White scale bars correspond to 100µm.(B) X-paint RNA-FISH as in Figure 5. (C) Analysis of allelic expression by pyrosequencing of genes relaxed from XCI in XaXe H9: GPM6B, lnc103, For each SNP, H9 genomic DNA (gDNA) and XaXe H9 and NSC cDNA were studied. The bar chart indicates the percentage of the peak height corresponding to each allele. (D) Allelic expression analysis of lnc121 during differentiation using sequencing. (E) Same as in (C) on POLA1 and COL4A6 genes. (F, G) Characterization of XaXe H9 hESC during undirected differentiation using RT-qPCR (F) and X-paint RNA-FISH (G). White scale bars correspond to 5µm.

A

XaXe

HUE

S1

d0 NSC

89%81%

NESTIN SOX2Xa

Xe H

UES1

NSC

94%90%

X-paint RNA X-paint RNA

B

7

C

E

lnc103GPM6B

0%

20%

40%

60%

80%

100%

gDNA XaXe H9

XaXe H9 NSC

% p

eak

heig

ht

0%

20%

40%

60%

80%

100%

gDNA XaXe H9

XaXe H9 NSC

H9 XaXe H9 NSCXaXe H9

G/A G/A A

lnc121

gDNA cDNA

Allele AAllele B

POLA1

0%

20%

40%

60%

80%

100%

gDNA XaXe H9

XaXe H9 NSC

COL4A6

0%

20%

40%

60%

80%

100%

gDNA XaXe H9

XaXe H9 NSCF

90%

XaXe

H9

65%

d0 d5

0

0,1

0,2

0,3

0,4

c2d0 c2d5 c3d0 c3d5

NANOG

0

0,2

0,4

0,6

c2d0 c2d5 c3d0 c3d5

OCT3/4

0

0,1

0,2

0,3

0,4

c2d0 c2d5 c3d0 c3d5

NANOG

0

0,2

0,4

0,6

c2d0 c2d5 c3d0 c3d5

OCT3/4OCT3/4 NANOG

Expr

essio

n re

lativ

e to

G

APDH

d0 d5 d0 d5Expr

essio

n re

lativ

e to

G

APDH

GX-paint RNA X-paint RNA

0

0,2

0,4

0,6

0

0,2

0,4

0,1

0,3

D

% p

eak

heig

ht

Allele AAllele B

Figure S6: XACT coating precedes the loss of XIST coating and H3K27me3 accumulation on the Xi (Related to Figure 6). (A) Quantification corresponding to experiment in Figure 6C, where XACT is in red and XIST in green. The barplot displays the percentage of nuclei (n>100) with corresponding expression pattern for passage 29 and 38. Number of nuclei were compared using a Fisher’s exact test.(B) Immunofluorescence coupled to RNA-FISH to simultaneously detect H3K27me3 (green) and XACT (red) in WIBR2 nuclei.(C) RNA-FISH for XIST (green) and XACT (red) in H9 cells shows co-accumulation of XIST and XACT on one X chromosome in 10% of the cells.

XaXi

H9

p.36

100%

merge

10%

XACTXIST

10%

% n

ucle

i

0%20%40%60%80%

100%

wibr2 p29 wibr2 p38

A

B

8"

100% 42% 42%p33 p33 p33WIB

R2 5

%O

2 XACTH3K27me3 merge

C

p=0.004

Cellular State XCI status Cell line Dataset H3K9me3 Dataset H3K27me3 Dataset INPUT Dataset H3K4me3 to

determine XCI status

hESCs XaXi H9 GSM1528888 GSM1528885 GSM1528891

hESCs XaXe H9 GSM1528889 GSM1528886 GSM1528892

hESCs XaY H1 GSM1003585 GSM733748 GSM733770

hESCs XaXi HUES6 GSM669886 GSM669887 GSM669888 GSM669889

hESCs XaXi HUES48 GSM772799 GSM772766 GSM772755 GSM772797

hESCs XaXe WIBR3 GSM1272771 GSM1272769 GSM1272772 GSM1272768

primary diff. cells XaXi IMR90 (Vallot, Ouimette et al.) GSM1528890 GSM1528887 GSM1528893

primary diff. cells XaXi IMR90 (Ren Lab) GSM521914 GSM521889 GSM521931/GSM469968

primary diff. cells XaXi Adipocyte GSM669968 GSM669930 GSM669934

primary diff. cells XaXi Colonic Mucosa GSM621668 GSM621673 GSM621669

primary diff. cells XaY AG04450 GSM1010914 GSM1010913 GSM94516

primary diff. cells XaY Duodenum Mucosa GSM621395 GSM621460 GSM621406

primary diff. cells XaY Duodenum Muscle GSM772909 GSM772840 GSM772910

primary diff. cells XaY Pancreas GSM537686 GSM537658 GSM537659

immortalized diff. cells XaXi hTERT RPE1 DRR003585 DRR003586 DRR003583

immortalized diff. cells XaXi vHMEC GSM613875 GSM669594 GSM613892

9

Table S1: Public ChIP-seq datasets analyzed, related to Figure 1, 2, S1 and S2.

Table&S2:&X+linked&genes&carrying&SNP&(n=78),&related&to&Figure&3Chr GeneStart GeneEnd GeneName StatuschrX 2746829 2800859 GYG2 escapeechrX 2852699 2886286 ARSE escapeechrX 3735569 3761898 RP11A706O15.1 escapeechrX 5758678 6146904 NLGN4X monoAallelicchrX 8496915 8700227 KAL1 monoAallelicchrX 9431335 9687780 TBL1X monoAallelicchrX 9754496 9917483 SHROOM2 monoAallelicchrX 11129421 11141198 HCCS monoAallelicchrX 11136239 11683821 ARHGAP6 monoAallelicchrX 12993227 12995346 TMSB4X reactivatedchrX 13789150 13956757 GPM6B reactivatedchrX 15337573 15353676 PIGA monoAallelicchrX 15843929 15873054 AP1S2 escapeechrX 16606126 16731059 CTPS2 escapeechrX 16737755 16783459 SYAP1 escapeechrX 16804550 16862642 TXLNG escapeechrX 16857406 16888537 RBBP7 escapeechrX 18443703 18671749 CDKL5 monoAallelicchrX 21958691 22025798 SMS monoAallelicchrX 23352133 23422489 PTCHD1 escapeechrX 23801290 23804343 SAT1 reactivatedchrX 23851470 23926057 APOO reactivatedchrX 24001837 24045303 KLHL15 monoAallelicchrX 24483338 24557954 PDK3 reactivatedchrX 24712036 25015103 POLA1 reactivatedchrX 38660685 38665790 MID1IP1 monoAallelicchrX 39909068 40036582 BCOR monoAallelicchrX 40507558 40595110 MED14 monoAallelicchrX 41192651 41223725 DDX3X monoAallelicchrX 41374187 41782716 CASK monoAallelicchrX 44732757 44971847 KDM6A monoAallelicchrX 46464753 46618490 SLC9A7 monoAallelicchrX 53401070 53449677 SMC1A monoAallelicchrX 53559057 53713673 HUWE1 monoAallelicchrX 57002803 57021970 SPIN3 monoAallelicchrX 57313139 57515629 FAAH2 monoAallelicchrX 65382391 65488709 HEPH monoAallelicchrX 65815479 65859108 EDA2R monoAallelicchrX 68835911 69259319 EDA monoAallelicchrX 69509879 69640682 KIF4A monoAallelicchrX 69664711 69725337 DLG3 monoAallelicchrX 70752933 70795747 OGT monoAallelicchrX 71401203 71522776 PIN4 monoAallelicchrX 71424510 71458897 ERCC6L monoAallelicchrX 78615881 78623164 ITM2A monoAallelicchrX 80457442 80554046 SH3BGRL monoAallelicchrX 85116185 85302566 CHM monoAallelicchrX 92925929 92928567 NAP1L3 escapeechrX 95939662 96859996 DIAPH2 reactivatedchrX 99839799 99854882 TNMD monoAallelicchrX 99883667 99894988 TSPAN6 monoAallelicchrX 100075384 100095921 CSTF2 monoAallelicchrX 100877787 100882833 ARMCX3 monoAallelicchrX 102930424 102943086 MORF4L2 reactivatedchrX 102995140 103055964 lnc103/PLP1 reactivatedchrX 106307650 106362057 RBM41 reactivatedchrX 106366657 106449670 NUP62CL reactivatedchrX 107386780 107682727 COL4A6 reactivatedchrX 107683074 107940775 COL4A5 reactivatedchrX 107975712 107979651 IRS4 reactivatedchrX 110187513 110470589 PAK3 reactivatedchrX 112859587 113181506 lncXACT reactivatedchrX 114795501 114885181 PLS3 reactivatedchrX 114957297 114959383 RP1A241P17.1 reactivatedchrX 115005825 115026519 linc115 reactivatedchrX 115033604 115085422 RP11A761E20.1 reactivatedchrX 117480036 117583924 WDR44 monoAallelicchrX 117629861 117820126 DOCK11 monoAallelicchrX 119384607 119392253 ZBTB33 monoAallelicchrX 119392505 119445411 TMEM255A monoAallelicchrX 121352438 121385472 lnc121 reactivatedchrX 122318006 122624766 GRIA3 monoAallelicchrX 129263337 129299861 AIFM1 monoAallelicchrX 133699868 133898352 PLAC1 reactivatedchrX 135579238 135594505 HTATSF1 monoAallelicchrX 137713735 138304939 FGF13 monoAallelicchrX 149934810 150067289 CD99L2 monoAallelicchrX 150564987 150577836 VMA21 monoAallelic

10

! 11!

****II.*Supplemental*Experimental*Procedures*

Production*of*homogeneous*populations*of*hESCs*regarding*their*XCI*status.*

We!had!initially!observed!in!our!H9!and!HUES1!original!cell!populations!by!immunofluorescence!and!

RNA1FISH!that!the!XCI!status!was!mixed,!with!cells!positive!and!cells!negative!for!H3K27me3!and!XIST!

co1existing.!These!statuses!are!overall!propagated!in!a!clonal!manner,!as!most!ES!colonies!contained!

either!XIST1positive! cells! or!XIST1negative! cells,! very! few! displayed! a!mixed! XCI! pattern.! Individual!

colonies! were! thus! manually! isolated,! fragmented! and! separated! in! an! amplification! plate! and! a!

corresponding! plate! dedicated! to! immunofluorescence! and! RNA1FISH! analysis.! Each! colony! was!

assigned! a! XIST+! or! XIST1! status! using! XIST! RNA1FISH,! which! was! further! verified! by!

immunofluorescence! with! an! anti1H3K27me3! antibody,! as! this! mark! is! strictly! dependent! on! XIST!

coating! in! mouse! (Kohlmaier! et! al.,! 2004)! and! in! human! (Silva! et! al.,! 2008).! Whereas! XIST+! cells!

(referred! to!as!XaXi)!have! clearly!undergone!XCI,!XIST1! colonies! could! in! theory!be! in!either!a!pre1!

(XaXa)! or! a! post1inactivation! (XaXe)! status,! although! pre1inactive! hESC! are! rare! and! tend! to!

spontaneously! undergo! XCI! (Makhlouf! and! Rougeulle,! 2011).! To! discriminate! between! these! two!

configurations,!we!performed!differentiation!of!cells! to!monitor!XIST!expression,!as!XIST! is! induced!

upon!differentiation!in!XaXa!cells!but!not!in!XaXe!cells!(Lengner!et!al.,!2010;!Mekhoubad!et!al.,!2012;!

Silva! et! al.,! 2008).With! this! protocol,! we! could! isolate! XaXi! and! XaXe! pure! populations.! XaXi! pure!

populations!maintained!their!XCI!status!for!at!least!10!passages!before!XaXe!cells!could!be!detected.!!

hESC*differentiation*

For! neural! differentiation,! hESCs! were! manually! dissociated! from! feeder! cells! and! plated! in!

suspension! during! 6! hours! in! KSR!medium! (DMEM/F12! supplemented!with! 20%!Knock1Out! Serum!

Replacement,!1%!Glutamax,!1%!non1essential!amino!acid!and!0.1%!β1mercaptoethanol,!all!from!Life!

! 12!

Technologies).! Formed!embryoid!bodies!were! transferred!on!poly1ornithine/laminin1coated!culture!

dishes! in! medium! containing! DMEM/F12! and! neurobasal! supplemented! with! N2! and! B27! (Life!

Technologies),!0.55mM!β1mercaptoethanol,!human!recombinant!noggin!(300ng/ml;!Preprotech)!and!

SB431542! (20μM;! Tocris).! Neural! rosettes! appeared! after! 8110! days! of! differentiation! and! were!

manually! transferred! in!N2B27!medium! supplemented!with! hEGF! (10ng/ml;! R&D! Systems),! hFGF2!

(10ng/ml;!PeproTech)!and!human!brain1derived!neurotrophic!factor!(hBDNF!10ng/ml;!R&D!Systems).!

After! 213! enzymatic! passages,! homogeneous!population!of! neural! stem!cells! (NSC)!were!obtained.!

NSCs!were! cultured! for! 3–5! days! until! confluence! and! passaged! (dilution! factor! about! 1:3! to! 1:5)!

using! 0.05%! trypsin/EDTA.! RNA!was! extracted! and! cells!were! fixed! at! day! 0,! 3,! 6,! and! 9! of! neural!

differentiation!and!from!NSCs.!NSCswere!characterized!by!immunocytochemistry!using!a!SOX2!rabbit!

antibody! (481400,! Life! Technologies,! dilution! 1/500)! and! NESTIN! mouse! antibody! (AB5922,!

Chemicon,!dilution!1/1000).!Anti1rabbit!Alexa1488!(A21206,!Life!Technologies)!and!anti1mouse!alexa1

568!(A10037,!Life!Technologies)!secondary!antibodies!were!used!at!1/1000!dilution.!Immunostaining!

was! quantified! using! the! colocalizationbioapplication! of! the! Cellomics! Array! Scan! VTI! HCS! Reader!

(Thermofisher! Scientist)! with! the! 10X! objective.! DAPI! staining! was! used! in! the! first! channel! for!

autofocus! and! nuclei! identification,! based! on! intensity! thresholds,! background! correction! and!

segmentation!parameters.!In!each!well,!50011000!nuclei!were!analysed!for!SOX2!and!NESTIN!staining!

and! positives! cells! were! defined! as! nuclei! expressing! a! minimal! intensity! and! area! of! the!

corresponding!target.!

Undirected!hESC!differentiation!was!obtained!after!manual! cutting!of!hESC!colonies!and!plating!of!

colony! fragments! on! gelatin1coated! plates! in! DMEMF112! medium! supplemented! with! 10%! FBS,!

0.1mM!NEAA!and!2mM!L1glutamine.!RNA!was!extracted!and!cells!were!fixed!for!RNA1FISH!at!day!0!

and!5!of!differentiation.!

ChIP@seq*data*analysis*

! 13!

In1house! generated! or! publicly! available! fastq! files!were! aligned! to! the! reference! human! genome!

hg19/GRCh37!with!bowtie!(Langmead!et!al.,!2009)!(1t!1q!1p!8!1S!1n!2!1e!70!1l!50!11maxbts!125!1k!1!1m!1!

11! phred331quals).! All! datasets! used! in! this! study! are! listed! in! Table! S2.! Aligned! reads!were!down1

sampled!to!the!minimum!available!read!count!for!each!sample!type!using!SAMtools(Li!et!al.,!2009):!

H3K27me3!ChIP1seq! (12.1M!reads),!H3K9me3!ChIP1seq! (21.9M!reads)! and! input! (11.1M! reads).!All!

subsequent!analyses!were!conducted!with!R!3.0.1!(www.r1project.org/).!We!assessed!the!global!XCI!

status!of!the!HUES6,!HUES48!and!WIBR3!lines!using!the!H3K4me3!ChIP1seq!datasets!(see!Table!S2).!

More!specifically,!we!studied!the!H3K4me3!enrichment!over!the!XIST!promoter,!which!is!indicative!of!

active!transcription!of!the!XIST!gene.!HUES6!and!HUES48!lines!displayed!H3K4me3!enrichment!over!

the!XIST!promoter,!we!therefore!classified!them!as!mainly!XaXi.!WIBR3!were!classified!as!XaXe!as!we!

could!not!detect!any!H3K4me3!enrichment!at!the!XIST!locus.!

To!create!a!chromosomal!ChIP1seq!profile,!we!divided!the!genome!into!100kbbins!and!calculated!the!

number!of!reads!per!million!of!mapped!reads!(RPM)!for!each!bin!using!bedtools!(Quinlan!and!Hall,!

2010).!The!log2!enrichment!value!for!each!bin!was!defined!as!the!log2!ratio!of!the!RPM!in!the!ChIP!

fraction! to! the!RPM! in! the! input! fraction.! For! zooms,!we!used!10kb!bins.!We! computed!Pearson's!

correlation! scores! between! log2! enrichment! ratios! (100kb! bins)! over! the! X! chromosome! and!

associated!p1values!using!a!randomization!of!the!data!sets!(n=106!permutations).!We!observed!a!high!

correlation!for!H3K27me3!ChIP1seq!profiles!(r=0.87,!p<1016)!but!our!H3K9me3!profile!slightly!differed!

from!the!published!one!(r=0.69,!p<1016),!probably!due!to!differences!in!the!specificity!of!the!antibody!

used.!

Discretization! was! done! using! jahmm! (Filion! and! Cusco,! http://arxiv.org/pdf/1405.5467.pdf)!

implemented!with!500bp1step!wiggle! file! for!H3K27me3!and!1000bp1step!wiggle! file! for!H3K9me3.!

For!chromosome1wide!representation,!we!computed!a!peak!density!score!over!100kb!bins:!number!

of!peaks/number!of!available!bins.!We!generated!occupancy!percentage!(Figure!1C),!calculating!for!

each!chromosome!the!cumulative!size!of!all!peaks!over!the!chromosome!size.!!

! 14!

We!used!GAT! (Heger!et!al.,!2013)! to!perform!genomic!association!analysis!of!our!peaks!with!gene!

annotation,!using!the!mappable!hg19!genome!as!the!workspace!and!the!ensembl!gene!annotation!as!

reference!annotation.!We!used!ngs.plot! (Shen!et!al.,!2014)! to!compute!average!maps!of!H3K9me3!

and!H3K27me3!log2!enrichment!over!gene!body!of!relaxed,!escapee!and!mono1allelic!genes.!

To!compare!H3K9me3!and!H3K27me3!profiles!over!the!X!chromosome!between!all!datasets!(Figure!

2G1H),! we! calculated! a! correlation! matrix! for! each! chromatin! mark,! composed! of! computed!

Pearson's!correlation!scores!between!log2!enrichment!ratios!over!the!X!chromosome.!!We!used!the!

corrplotR! package(Friendly,! 2002)to! graphically! display! this! correlation! matrix,! and! to! perform!

hierarchical! clustering! (method="ward")! and! subsequent! reordering! of! the! correlation!matrix.!We!

represented!the!obtained!clusters!by!black!rectangles!over!the!reordered!correlation!matrix.!

Identification*of*H9*informative*genomic*SNPs*

We! used! a! whole! genome! sequencing! datatset! for! H9! to! first! identify! genomic! SNPs! along! the! X!

chromosome,!which!could!be!informative!for!allelic!assessment!of!expression!(GSM1227088).!Reads!

were!aligned!with!bowtie!(Langmead!et!al.,!2009),!PCR!duplicates!were!filtered!out!using!SAMtools!

(Li! et! al.,! 2009)! and! reads! were! further! processed! using! GATK! (McKenna! et! al.,! 2010)! for! SNP!

identification! with! an! allelic! ratio! of! 0.5.! We! kept! high! confidence! SNPs! referenced! in! the!

dbsnp_137.b37! database! with! an! allelic! ration! of! 0.5! (n=34,378! for! X! chromosome).! We! then!

selected!all!genomic!positions!on!the!X!chromosome!that!were!covered!by!10!or!more!reads!in!both!

XaXi! and! XaXe! RNA1seq! data.! Pileups! for! each! RNAseq! dataset! were! generated! using! SAMtools!

mpileup!(Li!et!al.,!2009).!This!approach!allowed!us!to! identify!198!positions!on!the!X!chromosome,!

corresponding!to!78!genes,!for!which!we!could!measure!the!allelic!balance!in!XaXi!versus!XaXe!cells.!

Gene@specific*RNA*and*DNA@FISH**

FISH! experiments! were! performed! with! cells! cultured! on! 12mm! gelatin1coated! coverslips,!

permeabilized! in! CSK! buffer! supplemented! with! 0.5%! Triton! (Sigma1Aldrich),! 2mM! EGTA! (Sigma1

! 15!

Aldrich)! and! 2mM! VRC! (New! England! Biolabs)! for! 5min,! fixed! at! room! temperature! in! 3%!

Paraformaldehyde!(Electron!Microscopy!Science)/PBS!for!10min!and!washed!3!times!in!ice1cold!70%!

Ethanol.! Alexa4881labeled! probes! were! generated! by! nick! translation! for! XIST! (a! 10kb! fragment!

corresponding!to!XIST!exon!1,!gift! from!E.!Heard),!XACT! (RP11135D3,!BACPAC),!ATRX! (RP11142M11,!

BACPAC)! and! POLA1! (RP1111104L9,! BACPAC).! For! RNA! and! DNA1FISH,! all! probes! generated! from!

BACs!were!precipitated!with!human!Cot11!DNA!(Life!Technologies)!and!the!XIST1probe!with!Salmon!

Sperm!DNA! (Life! Technologies),! resuspended! in! 50%! Formamide/50%!Hybridization! Buffer! (4XSSC,!

20%!Dextran!Sulfate,!2mg/ml!BSA,!2mM!VanadylRibonucleoside!Complex)!and!denatured!for!7min!at!

75°C.! Cot11! precipitated! probes! are! additionally! pre1incubated! 15min! at! 37°C.! For! RNA1FISH,!

coverslips!were!dehydrated! in!90%!and!100%!ethanol!and! incubated!overnight!with!probe!at!37°C.!

After! three!50%!formaldehyde/2XSSC!washes!and! three!2XSSC!washes!at!42°C! for!4min,!coverslips!

were!mounted!in!Vectashield!plus!DAPI.!!

Microscopy*and*image*analysis*

All! images,! except! for! successive! RNA/DNA! FISH! analysis! (Figure! 4),!were! taken! on! a! fluorescence!

microscope!Axioplan!2!Imaging!(Zeiss)!with!a!cooled!Coolsnap!camera!(Roper!Scientifics)!controlled!

by! the! Metamorph! 7.04! software! (Roper! Scientific)! using! a! Plan1neofluar! 100X! oil! objective!

(numerical! aperture! 1.3,! Zeiss).! Optical! Z1sections! were! collected! at! 0.5µm! steps! through! each!

nucleus! at! different! wavelengths! depending! on! the! probes! used! (DAPI! [360nm,! 470nm],! FITC!

[470nm,! 525nm],! cy3! [550nm,! 570nm],! Texas! Red! [596nm,! 612nm]! and! cy5! [647nm,! 668nm]);!

approximately!15!optical! sections!per!nucleus!were! collected.! Stacks!were!processed!using! ImageJ!

1.46! (Abramoff! et! al.,! 2004),! and! throughout! the! manuscript! the! 3D1FISH! experiments! are!

represented! as! a! 2D1projection! of! the! stacks! (maximum! projection).! For! the! RNA/DNA1FISH!

experiment!with!cy3!(yellow)!and!FITC!(green)!labeled!chromosome!paints,!images!were!taken!using!

a!motorized!stage!1!DMI16000!inverted!microscope!(Leica),!with!CCD!Camera!HQ2!(Roper!Scientifics)!

controlled!by!the!Metamorph!7.04!software!(Roper!Scientifics)!using!a!HCX!PL!APO!100X!oil!objective!

! 16!

(numerical!aperture,!1.4,!Leica).!For!the!segmentation!and!volume!analysis!in!Figure!6,!we!used!the!

ImageJ!plugin!3D1suite!(Ollion!et!al.,!2013).!

RNA*extraction*and*RT@qPCR*

Total!RNA!was!extracted!from!all!cells!using!trizol.!RNA!was!treated!with!Turbo!Dnase(Invitrogen)!to!

remove! DNA! contamination.! One! μg! of! total! RNA! was! used! for! reversetranscription,! using! the!

SuperScriptVILO!cDNA!synthesis!kit! (Invitrogen).!mRNA!expression! levels!wereevaluated!using! real1

time! quantitative! PCR! (RT1qPCR)!with! the! SYBR!Green! kit! on! an! ABIPRISM!7900! real1time! thermal!

cycler! (Applied! Biosystems).! All! samples! were! run! induplicate.! Primers! sequences! are! XIST! (Fwd,!

ACATGCCTGGCACTCTAGCA;! Rev,! AAACATGGAAATGGGTAAGACACA),! OCT3/4! (Fwd,!

CCCCTGGTGCCGTGAAG;! Rev,! CTCGAGTTCTTTCTGCAGAGCTT),! NANOG(Fwd,!

CATGAGTGTGGATCCAGCTTG;! Rev,! CCTGAATAAGCAGATCCATGG)! and! GAPDH9 (Fwd,!

GAGTCAACGGATTTGGTCGT;!Rev,! TTCCCGTTCTCAGCCTTG.!RNA!expression! levels! for! the! transcripts!

ofinterest!were!normalized!against!the!reference!gene!GAPDH9according!to!the!21ΔCt!method.!

! 17!

!III.*Supplemental*References*

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