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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