supplemental experimental proceduresgenesdev.cshlp.org/content/suppl/2017/12/21/gad...supplemental...

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.

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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

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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

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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

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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

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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


hESCs_H9 iPSCs_EC-11 D

MIXL1 T EOMES

0

0.2

0.4

0.6

0.8

1

1.2

1.4


YAP

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 YAP


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


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


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)


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


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


0

1

2

3

4

5

6

7

8

9

0

2

4

6

8

10

12

14

Rel

. mR

NA

leve

ls

WNT3


hESCs_H9

iPSCs_EC-11

WNT3

Rel

. mR

NA

leve

ls

B

12


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


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)


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



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

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tion

prot

ein

bind

ing

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elop

men

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on

regu

latio

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hea

rt ra

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ll cy

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uscl

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sue

Dila

ted

card

iom

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Hyp

ertro

phic

car

diom

yopa

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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).

conchiestaras

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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

conchiestaras

Cross-Out

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

supplemental experimental proceduresgenesdev.cshlp.org/content/suppl/2017/12/21/gad...supplemental...

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