Estarásetal.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
hESC culture
H1 and H9 hESCs and EC-11 iPSCs were cultured in mTeSR1 media on Matrigel-
coated tissue culture plates. For colony expansion, Dispase was added to the plate for 5
minutes and after washes with DMEM-F12, colony fragments were split 1:3 ratio.
Medium was replaced daily. For differentiation experiments, colonies were
disaggregated into single cells at 1:6-1:8 split ratios following treatment with Accutase in
the presence of Rock inhibitors.
ChIP-qPCR and ChIP-seq
For the ChIP experiments, 3 × 106 hESCs were double-crosslinked with 0.2 mM di (N-
succinimidyl) glutarate (DSG, Sigma, 80424) 45 min followed by 1% formaldehyde 15
min. Following sonication, the cell lysate was incubated with antibody overnight at 4°C.
Magnetic beads were used to capture the immunocomplexes. The DNA was purified
using the Qiaquick PCR purification kit (QIAGEN, 28106) and eluted in 75ul of water and
2ul was used for each qPCR reaction. For ChIP-qPCR amplified material was detected
using SYBR green master mix (Life Technologies) on an ABI7300 (Applied Biosystems)
thermo-cycler. The obtained ChIP signal was normalized and shown as percentage of
Input. All primers used are listed in Supplemental Table 6.
Buffers: Lysis/IP buffer: 0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM
Tris pH 8, add fresh protease inhibitors. Wash buffer 1: 0.1% SDS, 0.1% NaDOC, 1%
Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM HEPES. Wash buffer 2: 0.1% SDS,
0.1% NaDOC, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 20 mM HEPES. Wash buffer
3: 0.25 M LiCl, 0.5% NaDOC, 0.5% NP-40, 1 mM EDTA, 20 mM HEPES. Wash buffer 4:
1 mM EDTA, 20 mM HEPES. Elution buffer: 1% SDS, 0.1 M NaHCO3.
For ChIP-seq experiments, at least 2 independent immunoprecipitations were carried out
and the eluted DNA from replicates was pooled before libray preparation. The ChIP
DNA was end repaired and 5′ phosphorylated using T4 DNA Polymerase, Klenow, and
T4 Polynucleotide Kinase (Enzymatics). Adaptor-ligated ChIP DNA fragments were
subjected to 15 cycles of PCR amplification using Q5 polymerase (NEB). AMPure beads
were used to purify DNA after each step (Beckman Coulter). ChIP fragments were
sequenced in an Illumina HiSeq 2500 sequencer.
1
Estarásetal.
mRNA extraction and Quantitative PCR
Total RNA was extracted using Quick RNA Zymo kit following manufacturer indications.
Then, 0.5 µg of total RNA was reverse transcribed using Transcriptor First Strand
Synthesis kit (Roche). The cDNA was amplified using SYBR green master mix (Life
Technologies) on an ABI7300 (Applied Biosystems) thermo-cycler. All results were
normalized to a RPS23 gene control. The ΔΔCt method was used to calculate relative
transcript abundance against an indicated reference. Unless otherwise stated, error bars
denote standard deviation between three biological replicates.
siRNA transfection
hESCs colonies were dissociated using Accutase and plated in presence of Rock
inhibitors at 1:8 ratio. 24h later, cells were transfected with Stemfect RNA transfection Kit
following instructions from the supplier. The siRNAs are listed in Supplemental Table 6.
Assays were performed 48 h after transfection.
Luciferase Assays
hESCs were plated into single cells using Accutase dissociation reagent. The next day,
Pgl3-derived constructs together were transfected using Lipofectamine 3000 reagent
(Invitrogen). Following 24h after transfection, hESCs were treated with the indicated
cytokines. Then, cells were lysed and processed following Dual-Luciferase® Reporter
Assay System Technical Manual (Promega). Luciferase activity was recorded in a 96-
well plate luminometer (Thermo Labsystems Luminoskan Ascent). The normalized
values from three independent biological replicates are shown in the graphs. Plasmids
used were described in Estaras et al, 2015.
Genetic manipulation of hESCs
Knock out cell lines were generating using the CRISPR/Cas9 vector pX458 (Addgene)
containing the sgRNAs to target the desired genes. sgRNAs sequences cloned are in
Supplemental Table 6. Briefly, hESCs were transfected with the mentioned vector and
48h later cells were GFP-sorted using cytometer. 10.000 GFP positive cells were plated
2
Estarásetal.
on irradiated fibroblast coated 10cm plates. After 10-15 days hESC clones emerged and
at least 30 clones were hand-picked and placed in 24 well plates. Positive KO clones
were identified by sequencing the gene of interest and confirmed by analyzing the
protein levels.
Doxycycline inducible YAP cell line was generated using a PiggyBac transposon system.
The Flag-YAP cDNA was cloned into the KA0717 vector (pPB-hCMV1-cDNA-
IRESVenus) and the given clone was then co-transfected into YAP-KO cells together
with transactivator and transposase-encoding vectors: pCAG-PBase (Austin Smith lab
(PMID: 19224983)) and KA0637 pPBCAG-rtTAM2-IN. 48h later, 50 µg/ml G418 was
added to select the transfected cells. After selection clones were isolated using the same
methodology explained above. Those clones with lowest leaking and robust Doxycicline
response were selected and used for experiments. The plasmids were kindly provided
by Dr. Kenjiro Adachi (Max Planck Institute for Molecular Biomedicine).
Indirect immunofluorescence for hESC and Cardiomyocytes
hESCs were fixed with formaldehyde 2% (FA) 10min and permeabilized with Triton 0.1%
for 10min. Day 20-30 cardiomyocytes were re-plated into slide chambers (Millipore
PEZGS0416) with RPMI/B27 media after Accutase dissociation. After at least 4
recovery days, cells were fixed using 4% FA 15 min. The next steps are common for
hESCs and cardiomyoctes; after fixation cells were incubated with blocking solution
(PBS Tween 0.1%, BSA 0.1%, FBS 10%) for 30min at room temperature. Primary
antibodies diluted in blocking solution were added overnight at 4°C. After washes,
secondary Alexa-conjugated IgG antibodies were added for 2h at room temperature.
Finally, mounting media containing DAPI and coverslip were added on the glass slides.
Images were captured by Zeiss LSM 780 confocal microscope using ZEN 2011
software.
Western Blot
For Western blotting, protein lysates were prepared for 20min on ice using RIPA buffer
with protease and phosphatase inhibitors. After centrifugation, supernatant containing
protein lysate were quantified using Bradford assays. SDS-PAGE electrophoresis using
10-30 µg of protein per sample and electroblotting were performed employing standard
3
Estarásetal.
procedures and equipment (BioRad). Trans-Blot Tubo Transfer System RTA transfer Kit
(BioRad) was used for transference to PVDF membranes. Primary antibodies
(Supplemental Table 6) were diluted in 0.5% BSA in PBS-Tween. HRP-conjugated
secondary antibodies and Super Signal West Pico chemiluminescent substrate (Thermo
#34078) were used for protein detection on BioRad Chemi-Doc Touch Detection System
device.
FACS analysis of Cardiomyocytes
For intracellular staining prior to FACS analysis, cells were dissociated into single cells
using digestion with Accutase for 15min. Cells were then pelleted, washed in PBS and
fixed with 1% FA 20min followed by 90% cold methanol 15min at 4 degrees. Then cells
were washed with FlowBuffer1 3 times and incubated with primary antibody in
Flowbuffer2 overnight (CtnT Lab Vision ms-295-p1, at 1:200). After two washes with
FlowBuffer2 cells were incubated 2h at room Temperature with the secondary antibody
(1:1000, Alexa 488 Goat anti-Ms IgG1,A-21121). After washes, cells were finally
resuspended in 500ul FlowBuffer1 and transfer into flow round-bottom tubes and
analyzed using The Becton-Dickinson LSR II flow cytometer. Percentages of CTNT-
positive cells were determined following pre-gating for intact single cells based on
appropriate settings for forward and side scatter in FACSDiva version 6.1.3 software.
FlowBuffer1: 0.5% BSA in PBS. FlowBuffer2: 0.5% BSA and 0.1% Triton in PBS.
GiWi protocol for Cardiomyocyte Differentiation
The GiWi protocol was developed by Lian et al in Nature Protocols, 2013. Briefly, H1
hESCs were cultured on Matrigel-coated plates until 80-90% confluence. Then, hESCs
were treated with GSK3i (12uM ChIR-99021, Selleck Chemical NC0466588, or 50nM
XV, Millipore 361558) for 24h in RPMI/B27-ins(minus insulin). At 72h, the Wnt inhibitor
IWP2 (7.5uM, Millipore 5.06072.0001) or XAV-939 (5uM, SelleckChem S1180) was
added to the media for 48h. Then, at day 5, fresh RPMI/B27-ins was added for another
48h. Finally, at day 7 media was changed to RPMI/B27 (containing insulin) and replaced
every 2-3 days. Robust contraction started around day 8-10.
Activin one step protocol
4
Estarásetal.
Yap-KO H1 hESCs were treated at day 0 with 100 ng/ml Activin A for 24h in TeSR
media. From day 1 on, RPMI/B-27 medium (with insulin) was used and replaced every
two days .The ratio of medium volume to cell numbers affects the efficiency of cardiac
differentiation. For optimal differentiation, use a volume of 4 ml per well of the 6-well
plate, 2 ml per well of the 12-well plate, and 1 ml per well of the 24-well plate. RPMI/B-27
medium was replaced every two days until day 7 of differentiation. From day 7, media
was replaced every three days. Robust spontaneous beating phenotype should occur
from day 9 onwards.
ChIP-seq analysis
ChIP fragments were sequenced in an Illumina HiSeq 2500 sequencer. Reads were
aligned to the Human hg19 genome assembly (NCBI Build 37) using STAR (v2.5.1b,
doi: 10.1093/bioinformatics/bts635. pmid:23104886) with default parameters. Reads
soft-clipping and splicing were turned off by specifying ‘--alignEndsType EndtoEnd --
alignIntronMax 1’ for ChIP-Seq mapping. Only reads that mapped uniquely to the
genome were considered for further analysis. Peak finding, motif finding and peak
annotation, genome browser read density files were performed using HOMER (v4.8,
http://homer.ucsd.edu/homer/, PMID: 20513432). Genomic binding peaks were identified
using the ‘findPeaks’ command in HOMER, with default settings of ‘-style factor’ for
transcription factors that usually have narrow peaks (Beta-catenin and Smad2.3) and ‘-
style histone’ for Pol II that usually have variable peak lengths covering large areas
(Ser7P, and all the other histone modification markers like H3K27ac etc.). Regions with
at least a fourfold enrichment over background input, a Possion p-value of 1e-4, and 25
normalized read counts were called as peaks. For the differential peaks analysis, peaks
were first merged by HOMER mergePeaks command with ‘-d given’ to look for literal
overlaps in peak regions and then the density of the merged peaks were compared by
HOMER getDifferentialPeaks. Peaks with at least a fold-enrichment of 2 (1.5 for RNAPII
CTD-Ser7P) and a Poisson p-value of 1e-4 were considered significant between
conditions. Peaks were assigned to gene targets based on the closest RefSeq defined
TSS by the annotatePeaks.pl command in HOMER. Overlapping peaks of YAP were
defined as the distance between the two peak centers smaller than 100 (for YAP peaks
between WT and YAP-KO) or 1000 bp (between YAP peaks and other markers like
TEAD4 and all histone markers like H3K27ac) by HOMER mergePeaks command.
HOMER makeBigWig.pl was used to generate the tracks for visualization. The ChIP-Seq
5
Estarásetal.
read densities were visualized along the genome using
IGV(http://www.broadinstitute.org/igv/, PMID: 22517427).
RNA-seq analysis
RNA-seq libraries were performed using stranded LT mRNA kit from Illumina and
sequenced in an Illumina HiSeq 4000 device. RNA-seq reads were mapped to the
human hg19 reference by STAR with default parameters. Only reads that mapped
uniquely to the genome were considered for further analysis. Gene expression levels
were calculated using HOMER by summing reads mapped across all gene exons of
RefSeq genes. The differential expression analysis was performed by edgeR (v3.16.1,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378882/) and genes with a false
discovery rate (FDR) < 0.05 were identified as differentially expressed between
conditions. PCA analysis was carried out using R ‘prcomp’ function on the DEseq2 rlog
(v1.14, https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8)
normalized raw counts. Plots were generated by R ‘ggplot2’ package (H.
Wickham.ggplot: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2009,
http://ggplot2.org). Gene ontology analysis of the β-catenin regulated genes was
performed using DAVID Bioinformatics Resources 6.8 version. Related Gene Ontology
terms were extracted from the Gene Ontology Consortium
(http://www.geneontology.org) by searching keywords of “heart”, “mesoendoderm
(mesoderm, endoderm, and primitive streak)”, and “pluripotency (pluripotency and
proliferation)”.Statistical analysis was performed by GAGE (v2.24,
https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-10-161) on the
expression changes of genes in each of the defined categories. P-values were
calculated based on a one-sided test to test for up-regulation and the Benjamini-
Hochberg method was used to correct for the multiple testing (Benjamini, Yoav;
Hochberg, Yosef (1995). Controlling the false discovery rate: a practical and powerful
approach to multiple testing. Journal of the Royal Statistical Society, Series B 57 (1):
289–300.). Heatmap was generated using R ‘gplots’ package (same as ChIP-Seq
analysis). Overlapping YAP peaks with transcription activity was carried out by looking
for peaks around the TSS of a DE gene within the range of +/- 50000 bp.
6
WT YAP -/- 0
0.005
0.01
0.015
0.02 TAZ
Rel
.mR
NA
leve
ls
A
B
0
10
20
30
40
50
GSK3i Act GSK3i Act
WT YAP-/-
EOMES
T
Rel
. mR
NA
leve
ls
0
5
10
15
20
25
30
0
1
2
3
4 SP5
Activin 100ng/ml
GSK3i (=ChIR 6µM)
LogFC GSK3i _WT vs UN_WT LogFC Activin_WT vs UN_WT LogFC GSK3i+Activin_WT vs UN_WT
LogF
C G
SK
3i_Y
AP
--/-
vs
UN
_WT
LogF
C A
ctiv
in_Y
AP
--/-
vs
UN
_WT
LogF
C G
SK
3i+A
ctiv
in_Y
AP
--/-
vs
UN
_WT
C
Estaras et al. FIGURE S1
Concentration Gradient:
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Rel
. mR
NA
leve
ls
MIXL1 T EOMES
siC siYAP Un Act Un Act
siC siYAP Un Act Un Act
hESCs_H9 iPSCs_EC-11 D
MIXL1 T EOMES
0
0.2
0.4
0.6
0.8
1
1.2
1.4
siC siYAP Un Act Un Act
YAP
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7 YAP
siC siYAP Un Act Un Act
7
A
0
50
100
150
200
Activin GSK3i
WT YAP-/-
MIXL1 -12Kb enhancer and promoter (SBS)-Luc
-12Kb - 0.1Kb
LEF/TCF SMAD
Luc
Rel
.Luc
.leve
ls
Activin GSK3i
- 0.1Kb
SMAD
Luc
0
50
100
150
200
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
WT β-cat-/-
siCtrl
untreated
siYAP siCtrl siYAP
Activin A, 50ng/ml 24h
Rel
. mR
NA
leve
ls
0 0.05
0.1 0.15
0.2 0.25
0.3 0.35
0.4 0.45
WT β-cat-/-
siCtrl siYAP siCtrl siYAP
β-cat-/- WT
β-catenin
DDX3
β-catenin-/- WT
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6 1.8
2 Oct4 mRNA
Rel
. mR
NA
leve
ls
β-cat-/- WT
MIXL1 EOMES
C B
MIXL1 promoter (SBS)-Luc
Estaras et al. FIGURE S2
8
NODAL
10Kb
EOMES
2.5Kb
[80]
[200]
[200]
[180]
[180]
[80]
[180]
[180]
[300]
[300]
[250]
[250]
"SMAD2,3 and β-catenin" differential peaks (±50Kb) in WT vs YAP-/- hESCs
1639
299
SMAD2,3 SMAD2,3 + β-catenin
209 (41.1%)
209 (8.84%)
B
WT: β-catenin
YAP-/- : β-catenin
WT: SMAD2,3
YAP-/- : SMAD2,3
WT: CTD-Ser7P RNAPII
YAP-/- : CTD-Ser7P RNAPII
+Act
ivin
A
WT: β-catenin
YAP-/- : β-catenin
WT: SMAD2,3
YAP-/- : SMAD2,3
WT: CTD-Ser7P RNAPII
YAP-/- : CTD-Ser7P RNAPII
+Act
ivin
SMAD2,3 + β-catenin β-catenin
Estaras et al. FIGURE S3
9
0 0.005
0.01 0.015
0.02 0.025
0.03 0.035
0
0.005
0.01
0.015
0.02
0.025
0.03
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
0
0.01
0.02
0.03
0.04
0.05
EOMES Regulatory Regions
0
0.01
0.02
0.03
0.04
0.05
0.06
0
0.05
0.1
0.15
OCT4 Regulatory Regions
Kb from TSS -44 -6 +1.2 -44 -6 +1.2
0
0.005
0.01
0.015
0.02
0.025
0
0.02
0.04
0.06
0.08
-1 NC
EOMES Regulatory Regions
-44 -6 +1.2 -44 -6 +1.2
Kb from TSS
OCT4 Regulatory Regions
% In
put
% In
put
C
β-catenin SMAD2,3 β-catenin SMAD2,3 ChIP:
β-catenin SMAD2,3 β-catenin SMAD2,3 ChIP:
-1 NC -1 NC -1 NC
siβ-catenin siSMAD2
siCtrl
YAP-/- hESCs (Activin)
Estaras et al. FIGURE S3
10
NXPH2
GCNT4
LHX8
ANXA1
AMOTL1
CYR61 CTGF
AFP VGLL3
AMOTL2
ANXA3
EIF1AY
ZNF662
BMP3
SHISA3 CTSF
NODAL
Log FC
-log1
0 p-
valu
e
Non DE
Diff expressed genes
RNA-seq WT vs YAP-/- hESCs
Estaras et al. FIGURE S4
842
130
YAP only 1323
YAP+H3K27ac
YAP+H3K27me3
C
Intergenic
Non conding -intron
Promoter
Exon
UTR
TTS
Distribution of YAP peaks in hESCs B
D
A
TEAD4 pvalue: 1e-496
POU5F1 pvalue 1e-90
TCF3 pvalue 1e-72
unknown ESC element /mESC-Nanog pvalue 1e-45
AP-1 pvalue 1e-44
Top Motifs bound by YAP in hESCs
41.5%
42.1%
11.8%
1.3% 1.7% 1.5%
11
0
0.02
0.04
0.06
0.08
0.1
0
0.01
0.02
0.03
0.04
0.05 0
0.02
0.04
0.06
0.08
0.1
Activin
Dox ng/ml GSK3i
0 0 0 0 20 20 20 50 50 50 ng/ml
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Rel
. mR
NA
leve
ls
MIXL1
EOMES
T
MIXL1
[190]
[190]
[190]
2.5Kb
EOMES
[190]
[190]
[190]
5Kb
T
[190]
[190]
[190]
5Kb
WT
YAP-/- minus Dox
YAP-/- plus Dox
CTGF
[170]
[170]
[170]
1.5Kb
D A
✓ ✓ ✓ ✓ Activin
GSK3i
IWP2
XAV ✓ ✓
✓ ✓
LEFTY1
0
0.005
0.01
0.015
0.02
A8301
✓ ✓ ✓ ✓
✓ ✓
✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗
✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗
✗ ✗ ✗
✗ ✗ ✗ ✗
Rel
. mR
NA
leve
ls
YAP-/- hESCs PiggyYAP hESCs C
CTD-Ser7P RNAPII ChIP-seq (+Activin)
WT
YAP-/- minus Dox
YAP-/- plus Dox
WT
YAP-/- minus Dox
YAP-/- plus Dox
WT
YAP-/- minus Dox
YAP-/- plus Dox
Estaras et al. FIGURE S5
0
1
2
3
4
5
6
7
8
9
0
2
4
6
8
10
12
14
Rel
. mR
NA
leve
ls
WNT3
siC siYAP Un Act Un Act
hESCs_H9
iPSCs_EC-11
WNT3
Rel
. mR
NA
leve
ls
B
12
Estaras et al. FIGURE S6
0
2
4
6
8 BAF60c
0
50
100
150 GATA4
Rel
. mR
NA
leve
ls
WT YAP-/-
72h treatment C
WT YAP-/-
B
Untreated Activin
LM and Cardiac developmental genes
LogF
C Y
AP
-/- /
WT
8 6 4 2 0 -2 -4
D
ME
SP
1 M
ESP1
/D
API
WT YAP-/-
+ A
ctiv
in
✓ ✗ ✗ ✗ ✗ ✗ ✗
✗ ✓ ✓ ✓ ✓ ✓ ✓
✗ ✗ ✓ ✗ ✗ ✓ ✓
✓ ✗ ✗ ✓ ✗ ✓ ✗
✗ ✗ ✗ ✗ ✓ ✗ ✓IWP2
XAV
WT YAP-/- WT-GiWi prot
GATA4
MESP1
Rel
. mR
NA
leve
ls
WT WT-GiWi prot
0
0.2
0.4
0.6
0.8
1
1.2
0
0.5
1
1.5
2
2.5
GSK3i
Activin
A8301
Day 5 Cardiac Precursor markers
0
2
4
6
0
10
20
30
Rel
. mR
NA
leve
ls
MIXL1 (WT)
MIXL1 (YAP-/-)
- Activin Inhibitor
+ Activin Inhibitor
D0 D1 D3 D5
CDK2
GATA6
BAF60c
EOMES
Cardiac inhibitors
YAP
T/BRACH
MSX1
CDX2
LM/Cardiac inductor
ME genes
D0 D1 D3 D5 Days of treatment
YAP-./- treated with Activin at D0 and Wnt inhibitor at D3
WT cells treated with GSK3i at D0
and Wnt inhibitor at D3
F
MIXL1 (WT)
MIXL1 (YAP-/-)
0
5
10
15
0
5
10
15
20
Gi Act
Rel
. mR
NA
leve
ls
G
E
H
Lateral meso
Cardiac meso
Cardio- myocytes
CDX2-
HAND1+
GATA4+ BAF60c+
MYH6+ TNNT2+
TBX5+
NKX2.5+
Cardiac prec. hESC
OCT4+ SOX2+
A
1 2 3 4 5 6 7
p=4e-5
p=0.76
13
CDX2 (+3.5Kb)
β-catenin RNAPII-Ser5P Smad2,3
Un Gi
% In
put
NC
Gi+ A.I WT hESCs
CDX2 (+3.5Kb)
NC CDX2 (+3.5Kb)
NC 0
0.005
0.01
0.015
0.02
0.025
0
0.1
0.2
0.3
0 0.5
1 1.5
2 2.5
3
Smad
β-cat CDX2 Smad β-cat MIXL1 EOMES..
Wnt and Activin interplay during lateral mesoderm induction
5Kb MIXL1
β-ca
teni
n Se
r7P-
RN
API
I
H1_untreated
H1_D1
H1_D3
H1_D5
H1_untreated
H1_D1
H1_D3
H1_D5
hESC-CM differentiation (D1 to D5) I
J
K
Estaras et al. FIGURE S6
14
CTNT positive cells
76.2% 0%
YAP-/-: Activin ONE STEP protocol
WT: Activin ONE STEP protocol
Neg control
Neg control
0% 0.2%
CTNT/FITC-A CTNT/FITC-A
CTNT/FITC-A CTNT/FITC-A
A
0
10
20
30
40
50
GiWi-prot
%C
TNT
posi
tive
cells
Wnt inhibitor added at Day 3
Without inhibitor
C Day 18 FACS analysis
(WT hESCs)
Estaras et al. FIGURE S7
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
1
2
3
4
5
6
7
8
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
2
4
6
8
10
12
14
Rel
. mR
NA
leve
ls
MESP1
siC siYAP Un Act Un Act
siC siYAP Un Act Un Act
hESCs_H9
iPSCs_EC-11
hESCs_H9
iPSCs_EC-11
MESP1 NKX2.5
NKX2.5
B
0.00E+00
1.00E-04
2.00E-04
3.00E-04
4.00E-04
5.00E-04
Z di
sc
card
iac
mus
cle
cont
ract
ion
stru
ctur
al c
onst
ituen
t of m
uscl
e sa
rcom
ere
orga
niza
tion
prot
ein
bind
ing
hear
t dev
elop
men
t ce
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D
FDR
Top significant GO terms from 3392 common UP-regulated genes in WT and YAP CM
15
Estarásetal.
Supplemental Figure 1. YAP Selectively Blocks Activin-mediated hESC
Differentiation to Mesendoderm. Related to Figure 1.
A. qPCR analysis of TAZ mRNA levels in WT and YAP-KO (YAP-/-) hESCs (Mean, n=3,
SD).
B. qPCR analysis of EOMES, T and SP5 mRNA levels in WT and YAP-KO (YAP-/-)
hESCs after 24h exposure to a concentration gradient of GSK3i and Activin molecules.
Above, the concentration of GSK3i and Activin used for the RNA-seq in Figure 1. (Mean,
n=2, SD).
C. A scatter plot of the log2 fold-change (LogFC) of the gene expression changes of
samples compared to the wild-type untreated (UN_WT) samples. The groups of genes
shown here are also presented in Figure 1D. LogFC were calculated by edgeR using
two replicates each group. Y-/- denotes YAP-KO cells, GSK3i and Activin denote the
treatment. Higher logFC was observed in YAP-KO cells compared to WT cells when
treated with Activin.
D. qPCR analysis of YAP, MIXL1, EOMES and T mRNA levels in control and YAP
siRNA transfected H9 hESCs and EC-11 iPSCs line (Mean, n=3, SD).
Supplemental Figure 2. β-catenin is Required for Activin-induced Differentiation
of YAP-KO hESCs. Related to Figure 2.
A. Graphs show normalized luciferase activity of transfected hESCs treated as indicated
below the graph (24h). The MIXL1 gene regulatory regions were assessed (see
captures on top) and two different constructs were tested: 1) SMAD plasmid, containing
the MIXL1 promoter and SMAD binding Site (SBS) at -0.3 Kb from TSS, and 2)
SMAD+LEF plasmid, which contains the SMAD and LEF-1 (-12Kb) sites. The graphs
plot Luciferase activity from three independent biological replicates.
B. Isolation of β-catenin-KO (β-cat-/-) hESCs. Left, immunoblot analysis of β-catenin
protein levels in WT hESCs and the knockout clone. Right, phase contrast microscopy
analysis of the morphology of WT and β-catenin-KO hESCs. The graph shows the qPCR
analysis of Oct4 mRNA levels in the WT and the KO cell lines (Mean, n=2, SD).
C. WT and β-catenin-KO hESCs were transfected with Control or YAP siRNAs and the
next day the cells were treated with Activin for 24h. The graphs show the mRNA levels
of MIXL1 and EOMES assessed by qPCR analysis after 48h of transfection (Mean, n=2,
SD).
Estarásetal.
Supplemental Figure 3. Activin Induces β -catenin Binding to ME and Wnt
Responsive Genes in YAP-KO hESCs. Related to Figure 3.
A. WT and YAP-KO hESCs were treated with Activin for 15h and ChIP-seq of SMAD2,3,
β-catenin and RNAPII CTD-Ser7P were performed. Captures show the distribution of the
immunoprecipitated proteins in NODAL and EOMES genes.
B. Venn diagrams show proximal SMAD2,3 and β-catenin differential peaks in WT
versus YAP-KO cells (50Kb range). For instance, the lower diagram shows that there
are 508 new β-catenin peaks in YAP-KO cells and among them, there are 209 peaks
that are close (50Kb range) to a new SMAD2,3 peak. These data indicate that almost
50% of new β-catenin peaks in YAP-KO cells are correlated with proximal recruitment of
SMAD2,3.
C. ChIP-qPCR analysis in Activin-treated YAP-KO cells transfected with siRNAs against
SMAD2 or β-catenin for a total of 48h. The immunoprecipitated proteins are indicated on
the top of the graphs. The regulatory regions analyzed are shown at the bottom of the
graphs (Mean, n=3, SD).
Supplemental Figure 4. YAP Binds Developmental Enhancers in hESCs. Related to
Figure 4.
A. Top YAP binding motifs in H1 hESCs identified by ChIP-seq.
B. A diagram shows the genomic distribution of YAP peaks in hESCs.
C. A graph shows the number of YAP peaks that co-localize with the repressive histone
mark H3K27me3 and the active enhancer mark H3K27ac in hESCs.
D. A volcano plot diagram showing differential expressed genes (in red) in WT versus
YAP-KO hESCs.
Supplemental Figure 5. YAP Repression of WNT3 prevents Premature
Differentiation in Response to Activin. Related to Figure 5.
A. The graph shows qPCR analysis of LEFTY mRNA levels in the YAP-KO cells (YAP-/-)
treated with specific inhibitors and cytokines as indicated below the graph. (Mean, n=3,
SD).
B. Graphs show qPCR analysis of WNT3 mRNA levels in control and YAP siRNA
transfected H9 hESCs and EC-11 iPSCs lines (Mean, n=3, SD).
Estarásetal.
C. PiggyYAP cells (see Figure 5F) were treated with Activin or GSK3i for 24h alone or
together with Doxycycline at indicated doses and the mRNA levels of the indicated
genes were analyzed by qPCR. (Mean, n=2, SD).
D) Genome browser captures of RNAPII CTD-Ser7P ChIP-seq in Activin-treated (15h)
WT hESCs and PiggyYAP cell line before and after Doxycycline treatment (24h).
Supplemental Figure 6. Activin Selectively Induces Differentiation to Cardiac
Mesoderm in YAP-KO hESCs. Related to Figure 6.
A. A scheme shows the differentiation stages from hESCs to beating cardiomyocytes.
Below, the main regulators of the differentiation process are indicated.
B. The boxplot shows the LogFC expression of Lateral Mesoderm and early cardiac
developmental genes in WT or YAP-KO hESCs in untreated and Activin-treated cells.
Adjusted p value for multiple testing corrections is shown above each box.
C. Graphs show the levels of cardiac mesoderm markers GATA4 and BAF60c. Specific
treatments are indicated below the graphs. The graphs show the average of two
representative experiments of at least 4 independent replicates. Mean (SEM; n=2).
D. MESP1 protein levels were analyzed by immunofluorescence in WT and YAP-KO cells
after 72h following 1 Day of Activin exposure.
E. Graphs show the levels of cardiac precursor markers GATA4 and MESP1. Specific
treatments are indicated below the graph. As a control, WT hESCs treated with GiWi
protocol (pink bar) is shown. Mean (SEM; n=3).
F. Immunoblot analysis of specific marker proteins. WT cells (left) were treated with the
GSK3i at Day 0 and Wnt inhibitor at Day3. YAP-KO cells (right) were treated with Activin
for 24h, followed by addition of the Wnt inhibitor at day 3. Cell extracts were obtained at
Day 0 (hESC), Day 1, Day 3, and Day 5.
G. qPCR analysis show mRNA levels of MIXL1 in WT and YAP-KO hESCs after
different concentrations of GSK3i (50nM to 5nM) and Activin (100ng/ml to 5ng/ml)
treatment for 24h. The graph shows the average of two representative experiments of at
least 4 independent replicates. Mean (SEM; n=2).
H. qPCR analysis show mRNA levels of MIXL1 in WT and YAP-KO hESCs after
treatment with GSK3i or Activin in presence or absence of Activin inhibitor A8301 (1µM).
The graphs show the average of two representative experiments of at least 4
independent replicates. Mean (SEM; n=2).
Estarásetal.
I. Genome browser captures show b-catenin and Ser7P-RNAPII distribution on MIXL1
gene in hESCs (D0) and at Day1, Day3 and Day5 after initial differentiation following the
GiWi protocol.
J. ChIP-qPCR analysis of β-catenin, SMAD2,3 and RNAPII-Ser5P binding to CDX2
enhancer (+3.5Kb) after treatment with GSK3i in presence or absence of Activin inhibitor
in WT hESCs. NC means negative control region. Mean (SEM; n=2).
K. A schematic depiction to summarize the interplay between Wnt and Activin signaling
pathways during lateral mesoderm induction.
Supplemental Figure 7. Human Cardiomyocyte Differentiation using a ONE-STEP
Protocol. Related to Figure 7.
A. EC-11 iPSCs and H9 hESCs were transfected with control or YAP siRNA. Next day,
transfected cells were treated with Activin for 24h. The graphs show qPCR analysis of
MESP1 and NKX2.5 mRNA levels at Day 5 after initial Activin treatment (Mean; n=3,
SD).
B. Representative flow cytometry plots of WT and YAP-KO cells stained with CTNT
antibody after 22 days from initial Activin treatment. The percentage of positive CTNT
cells are shown in the graphs. The negative controls lack primary antibody.
C. Following the GiWi protocol, WT hESCs were treated with GSK3i at Day 0 and then
left untreated (blue bars) or treated with the Wnt inhibitor (IWP2 7.5µM) at Day 3 (gray
bar). A graph shows the percentage of CTNT positive cells analyzed by FACS at Day 18
after initial differentiation.
D. A bar plot shows top enriched GO terms for the 3392 common up-regulated genes in
WT and YAP-KO derived cardiomyocytes.
Supplemental Video 1. YAP-KO hESCs differentiated into cardiomyocytes using the
Activin ONE STEP protocol. Day 14.
Supplemental Table 1. List of genes regulated in YAP-KO cells after Activin or
GSK3i treatment (24h and 72h) and list of genes regulated in the β-catenin and
YAP double KO cell line
Estarásetal.
Supplemental Table 2. List of YAP and TEAD peaks identified by ChIP-seq in
hESCs
Supplemental Table 3. List of genes regulated in YAP-KO versus WT hESCs
Supplemental Table 4. List of genes regulated in WT and YAP-KO cells after
Activin or GSK3i treatment (high concentration, XV50nM) (30h)
Supplemental Table 5. List of genes up-regulated in WT and YAP-KO
cardiomyocytes compared to hESCs
Supplemental Table 6. List of primers, antibodies, siRNAs and sgRNAs
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