SMAD2/3 ChIP

Motifs in EndoMotifs in hESC

% o

f pea

ks w

ith m

otif

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10

20

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50

60

SMAD FOXH1 SCA

Gene 10 kb(up) 10 kb(down) Intergenic Gene 10 kb(up) 10 kb(down) Intergenic

0

10

20

30

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SMAD FOXH1 SCA

Yoon_Supplemental Figure 1

A

B

hESC

Endoderm

ChIP-seq:SMAD2/3SMAD3SMAD4FOXH1

Motif Analysis-or-

PeakCalls

A

B

C Endoderm (1270 regions)

hESC (813 regions)

0 1E-02 1E-04 1E-06 1E-08

regions with SCA motif hESC, 556/813 Endoderm, 776/1270

regions without SCA motif hESC, 257/813 Endoderm, 494/1270

Functional enrichment with GREAT

all four TFs binding within 1 kb hESC, 813Endoderm, 1270

p value

0 1E-02 1E-04 1E-06 1E-08 1E-10p value

1 kb

vs

GO

Bio

logi

cal P

roce

ssG

O B

iolo

gica

l Pro

cess

regulation of cell migrationregulation of locomotion

mesoderm formationSMAD protein signal transduction

mesodermal cell di�erentiationorgan regeneration

mesoderm morphogenesisTGF-β receptor signaling pathway

endoderm formationendoderm development

gastrulationformation of primary germ layer

negative regulation of transcription endothelial cell morphogenesis

negative regulation of cell di�erentiation tissue morphogenesis

body morphogenesispattern speci�cation process

mesoderm morphogenesis

formation of primary germ layerembryonic pattern speci�cation

anterior/posterior pattern formationgastrulation

axis speci�cation

556 with SCA 257 no SCA

776 with SCA 494 no SCA

Yoon_Supplemental Figure 2

A

B

C

IgG HEB E2A FOXH1 Lys

+ SB

S2/3

- SB

WB:FOXH1

IgG HEB E2A FOXH1 LysS2/3

IP:

+ EtBr

- EtBr

pSMAD2

FOXH1

SMAD2/3

Tubulin

E2A

HEB

SMAD4

+ SB- SB

LEFTY

WB:FOXH1

IP:

Yoon_Supplemental Figure 3

heb MO e2a MO 1:4 A B

C

Yoon_Supplemental Figure 4

Ctrl MO

xbra xbra

Uninjected e2a MO mouse e2a mRNAe2a MO+

e2a mRNAxbra xbraxbra xbraxbra

sox17βsox17βsox17βsox17β

Supplementary Information for

HEB and E2A Function as SMAD/FOXH1 Cofactors

Se-Jin Yoon, Andrea E. Wills, Edward Chuong, Rakhi Gupta, and Julie C. Baker

Supplemental Figures

Supplemental Figure 1 (Supports Figure 1). (A) Flow chart of motif analysis

from ChIP-seq data obtained for SMAD2/3, SMAD4, SMAD3 and FOXH1 in

hESCs and endoderm. (B) Bar graphs show the genomic distribution of motif-

associated peak regions in SMAD2/3 ChIP in hESCs (hESC, left) and endoderm

(Endo, right) were classified into annotated genomic regions: Genebody (Gene),

10 kb upstream (up) promoter region from the 5’ genebody region, 10 kb

downstream (down) region from the 3’ genebody region, or intergenic region

using UCSC Known Genes (Human browser hg18).

Supplemental Figure 2 (Supports Figure 1). GO biological process of SCA

motif associated regions. (A) All four (SMAD2/3, SMAD3, SMAD4, and FOXH1)

transcription factors (TFs) binding regions within 1 kb window. Schematic

stratege for the comparison of SCA motif associated or not associated regions.

(B) Funcional enrichment with GREAT in hESCs compared to SCA motif

associated 556 regions with not associated 257 regions. (C) Funcional

enrichment with GREAT in endoderm compared to SCA motif associated 776

regions with not associated 494 regions. The GO term is indicated along the y-

axis, and the p value for the significance of enrichment for the top GO terms

along the x-axis.

Supplemental Figure 3 (Supports Figure 2 and 3). (A) Co-

immunoprecipitation (coIP) of endogenous FOXH1 with SMAD2/3 (S2/3), HEB,

E2A and FOXH1 in hESCs in the absence of EtBr (- EtBr) or in the presence of

EtBr (+ EtBr) at 400 µg/mL. Rabbit IgG was used for IP instead of antibody as a

negative control. Input lysates (Lys) was used 2% IP sample of total lysates. (B)

CoIP of endogenous FOXH1 with SMAD2/3 (S2/3), HEB, E2A and FOXH1 on

cells differentiated in Activin for 3 days and then treated cells for 48 h with Activin

in the absence of SB431542 (- SB) or in the presence of SB431542 (+ SB). (C)

Protein expression analysis using Western blotting during hESCs differentiation

into endoderm with Activin in the absence of SB431542 (- SB) or in the presence

of SB431542 (+ SB). Tubulin was used for loading control.

Supplemental Figure 4 (Supports Figure 5). (A) Injection of 20 ng of HEB MO

in both blastomeres at the 2-cell stage does not result in gross morphological

defects at gastrulation (stage 10.5) or in early tailbud embryos (stage 22). (B) Co-

injection of 10 ng of E2A MO with nuclear β-galactosidase mRNA in one

blastomere at the 4-cell stage results in cell-autonomous inhibition of blastopore

formation and of xbra expression. Red color represents red-gal substrate

staining and marks the injected portion of the embryo. (C) Embryos co-injected

with e2a MO and mouse e2a mRNA were compared to uninjected controls for

expression of xbra (mesoderm) and sox17β (endoderm). While e2a MO injection

led to downregulation of xbra, this was rescued by co-injection with mouse E2A

mRNA.

Supplemental Materials and Methods

Cell culture

H9 hESCs (WiCell) were maintained on mouse embryo fibroblast (MEF) feeder

layers in DMEM/F12 supplemented with 20% KnockOut serum replacement

(Gibco), 0.1 mM nonessential amino acids (Gibco),Glutamax (Gibco), 0.1 mM 2-

mercaptoethanol, and 8 ng/mL recombinant human fibroblast growth factor-basic

(bFGF; Peprotech). Cultures were routinely passed with 200 U/mL type IV

collagenase (Gibco) in 1:3 ratio every 4 days. For endoderm differentiation,

hESCs were maintained on plates coated with growth-factor-depleted matrigel

(BD Biosciences) in MEF-conditioned medium (CM) for 2 days. Endoderm

differentiation was performed in RPMI (Gibco) supplemented with

penicillin/streptomycin, 100 ng/mL recombinant human Activin A (R&D Systems)

and varying concentrations of defined FBS (HyClone) as 0% for the first 24 h,

0.2% for the second 24 h, and 2% for subsequent days of differentiation as

previously described (D'Amour et al., 2005; Pan et al., 2011).

Immunoprecipitation and Western blot analysis

Cells were harvested in lysis buffer (50 mM Tris pH 8.0, 150 mM Nacl, 1% NP-

40) supplemented with a cocktail of protease inhibitors (Roche). For co-

immunoprecipitation (coIP), 3 µg each antibody or rabbit IgG (R&D Systems)

coupled with Protein A beads (Invitrogen) was incubated overnight at 4°C. Beads

were washed five times with lysis buffer and eluted with SDS-loading buffer.

Proteins were separated on SDS polyacrylamide gels, transferred onto PVDF

membranes (Amersham) and detected through horseradish-peroxidase

conjugated secondary antibodies (Santa Cruz Biotechnololy) and

chemiluminescent ECL (Amersham) exposure. The primary antibodies used

were anti-GATA4, anti-GATA6, anti-SOX17 and FOXH1 from R&D Systems,

anti-SMAD2/3, HEB and E2A from Santa Cruz Biotechnologyand anti-LEFTY

and anti-Tubulin from Abcam as described in detail in our recent study (Kim et al.,

2011, in press). Nuclear and cytoplasmic extracts were isolated from derived

endoderm using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce)

based on the manufacturer’s instruction. Etidium bromide (EtBr) treatments were

performed as demonstrated earlier (Lai and Herr, 1992). EtBr (400 µg /mL) was

added and the lysates were incubated on ice for 30 min. The original

concentration of EtBr was maintained during the washing steps.

Chromatin immunoprecipitation (ChIP)

ChIP assays were carried out for hESCs and derived endoderm grown to a final

count of 4-5 x 106 cells for each reaction. Cells were chemically crosslinked with

1% formaldehyde for 15 min at room temperature and the reaction was stopped

by adding Glycine (to 125 mM) for 5 min. Cells were rinsed with PBS and

resuspended in lysis buffer (50 mM Tris pH8.1, 10 mM EDTA, 1% SDS) with

protease inhibitor cocktail (Roche), and fragmented with a Branson 450 Sonifier

(eight pulses at 40%) to a size range of 200 to 600 bp. Solubilized chromatin was

diluted 3-fold in ChIP dilution buffer and, after removal of a control aliquot,

immunoprecipitated with Protein A or G magnetic beads (Invitrogen) coupled with

5 µg of each antibody at 4°C overnight. Primary antibodies against SMAD2/3,

SMAD4 and FOXH1 from R&D Systems and SMAD3 from Abcam were used for

ChIP-seq and two different primary antibodies (a and b) against HEB and E2A

from Santa Cruz Biotechnology were used for the validation of binding to

SMAD/FOXH1 targets. Immune complexes were washed sequentially with Low

Salt Immune Complex Wash, High Salt Immune Complex Wash, LiCl Immune

Complex Wash, and TE, and then eluted in Elution Buffer (1% SDS, 0.1M

NaHCO3). After crosslink reversal and Proteinase K treatment, ChIP and control

DNA samples were extracted with phenol-chloroform and purified with QIAquick

PCR Purification Kit (Qiagen).

For sequential ChIP, crosslinked chromatin from hESCs was

immunoprecipitated with antibody against SMAD2/3 as described above except

that chromatin was eluted in a solution of 30 mM DTT, 500 mM NaCl, and 0.1%

SDS at 37°C. Eluted chromatin was diluted 10-fold, subjected to a second

immunoprecipitation with antibody against FOXH1, HEB or rabbit IgG, and then

eluted with standard Elution Buffer. The isolated DNA was extracted and purified

as above. The primers used for qPCR to quantify the ChIP-enriched DNA are as

shown in below. Relative occupancy values were calculated by determining the

apparent immunoprecipitation efficiency (ratio of the amount of

immunoprecipitated DNA to that of the input sample) and normalized to the level

observed at a negative control region (GAPDH intronic regions), which was

defined as 1.0.

.

Primers used for ChIP-qPCR (F, Forward; R, Reverse)

ANK2: F: CACCTACCTCATCATCTCTG, R: CCACCTGCTGTTATGTGTAT ETS2: F: GGATTCTGATGTCTTGAGTG, R: CTCTTCTGTCTGTTTTCCAG CYP2B6: F: AGATGTGGACACATTCATTC, R: GAGATCCAGAAGCACAGATA ZIC5: F: AATAGGCCTAGCTAGATGGT, R: GGGTCATCACAATAACAAAA MST150: F: GGTTCATCTGTGGTGAATAC, R: ATCATAGCAGATGTTCAAGG TDGF1: F: AGCTGGAGGGTACATACATA, R: ATCATAGGGCACTTAGGTCT LINGO1: F: CACCAAGCCAGTTTTATTAG, R: TGTGGACTCAAGCTATTAGG EOMES: F: TTTTGTTCTCCACCTACTTG, R: TTTCTGGACATCCAGTTAAG LEFTY1: F: TTGTAAACAGTCTCCCAATC, R: CTATTATCCCTCTGCAAACA LEFTY2: F: AGCCTGAAGAGTTTTGTTTG, R: TCCTGACGACTAATCAGACC LEFTY2(intergenic): F: CACAGACACTGAAATTCCTC, R: TGCTGAATATCCCATAACAT CER1: F: ACATGTTCCTCAACAGGATA, R: TCTTAGAATCCACGTTCTCA MIXL1: F: TTGGATTCAGGATGTGTTAG, R: GTCTGTCCTCATCAAAGCTA POU5F1: F: CTGACTTCAGGTTCAAAGAA, R: TGAGCACTTGTTTAGGGTTA GAPDH: F: AAGTGGATATTGTTGCCATC, R: GGAATACGTGAGGGTATGAA

Motif analysis and association of genes with each motif

The setting for peak-calling and sliding window size was 300 bp and the

threshold number of reads required for peak to be called was 11 reads. The false

discovery rate allowed was 0.01. Separate input library controls for hESCs and

endoderm were used to normalize enrichment calculations in CisGenome. To

discover novel motifs, we extracted the 2000 highest scoring peaks from each

dataset as determined by CisGenome. After extending each peak 100 bp in each

direction, we extracted the corresponding repeat-masked DNA sequence from

the hg18 genome downloaded from UCSC. We then used these sequences as

an input to MEME (v 3.5.4) with default settings and examined the results

manually. To search for previously characterized motifs similar to the SMAD

Complex Associated (SCA) motif, we used STAMP

(http://www.benoslab.pitt.edu/stamp/) and searched against the TRANSFAC (v

11.3) database. To determine which of the original SMAD complex targets

contain the SCA motif, we extended each peak 200 bp in both directions,

extracted the unmasked sequence from hg18, and used FIMO (v 4.3.0) to scan

for instances of the SCA motif with p < 0.0001. Overlapping target regions were

determined by requiring at least one SMAD2/3, SMAD3, SMAD4, and FOXH1

binding site to be present within a window of 1000 bp.

To determine the genomic composition of each dataset, we used the

UCSC knownGene annotations. For each dataset, we sequentially determined

overlap (at least 1 bp) with the gene body (broken down into exons and introns),

then proximal promoters (10 kb upstream the 5’ end of the gene), then

downstream regions (10 kb downstream the 3’ end of the gene), and all

remaining peaks were classified as ‘intergenic.’ Ontology enrichment for each

dataset was performed using GREAT (hg18, default settings, genome-wide

background, http://great.stanford.edu) (McLean et al., 2010).

Xenopus tropicalis embryo culture

Embryos were generated by natural mating as follows: adult female Xenopus

tropicalis were injected 18-30 h before embryo collection with 10 U human

chorionic gonadotropin (HCG) (Sigma). Four hours before embryo collection,

male and female frogs were injected with 100 U and 400 U of HCG, respectively,

and allowed to begin amplexus. 30-45 min after the onset of egg laying, embryos

were collected and dejellied in 1/9 MR+ 3% cysteine. Embryos were cultured in

1/9 MR at room temperature or at 28°C (generally, embryos analyzed at stage 11

or younger were reared at 28°C to facilitate earlier collection, while embryos

analyzed at later stages were reared at room temperature). Staging was

assessed according to (Niewkoop and Faber, 1967). For additional details see

(Khokha et al., 2002).

Microinjection

Morpholinooligonucleotide design and microinjection

Translation-blocking (e2a, heb) and splice-blocking (heb):

Morpholinooligonucleotides (Gene Tools, LLC) were designed against X.

tropicalis gene products, using the JGI genome browser and ESTs to identify

transcription start sites, intron/exon boundaries, and potential polymorphisms.

Unless otherwise noted, MOs were co-injected with 10 ng of fluoresceinated

standard control MO as a tracer for injection.

Morpholino oligonucleotide sequences used: e2a: TGTCATCCTCTGCTGTTGATTCATT heb (translation-blocking): CGGCCATCCGCTGCTGAGGATTCAT heb (splice-blocking): GACCCCGAAGGATATCGTACCGCAC

mRNA synthesis and injection

Linearized plasmid DNA was transcribed with Sp6 polymerase, using NTP mix

containing 5’ methyl-G cap (mmessagemmachine, Ambion). mRNA was

precipitated first with LiCl, then re-precipitated using ammonium acetate,

quantitated, resuspended and stored at -80°C. Injections were performed at

stages and with amounts specified in each experiment.

LacZ lineage tracing

100-200 pg mRNA encoding nuclear-localized β-galactosidase (nβgal-CS2+) was

injected alone or in conjunction with other mRNAs. At the desired stage, embryos

were fixed for 30 min in MEMFA, washed 3X in PBS+0.1% tween 20, then

incubated with Red-Gal substrate (Research Organics) at 37°C until well-

developed (Sive, 2000). Embryos were then washed with PBS+0.1% tween 20,

fixed 2 h in MEMFA, and dehydrated in methanol. In some cases, dehydrated

stained embryos were subsequently used for in situ hybridization.

Whole mount in situ hybridization

Embryos were developed to the desired stage and then fixed in MEMFA for 2-6 h

at room temperature or overnight at 4°C. X. tropicalis multi-basket in situ

hybridization protocols were followed as described in (Khokha et al., 2002).

Plasmids used for in situ hybridization probes:

Gene: Source: Vector:

xbra (Harland lab) CS107

sox17b (Harland lab) CS107

gsc IMAGE:7536652 pCS108

e2a IMAGE:7660124 pcmv-sport6

heb IMAGE:7679436 pcmv-sport6

RT-PCR

Injected or uninjected embryos (3 or 5 per experiment) were digested using 200

µl of RNA lysis buffer (5 mM EDTA, 50 mM Tris pH 7.5, 50 mM NaCl, 0.5% SDS,

250 µg/ml Proteinase K). Embryos were digested for 1 h at 42°C,

phenol/chloroform extracted and precipitated overnight at -20°C with ethanol and

sodium acetate. Pellets were resuspended in nuclease-free water, treated with

DNAse I for 1 h at 37°C, phenol/chloroform extracted again, and RNA

precipitated with ammonium acetate and ethanol. RNA pellets were resuspended

in nuclease-free water, random hexamers added, and reverse transcription was

carried out using SuperScript III reverse transcriptase (Invitrogen). Where

possible, primers were designed to span a small intron to facilitate detection of

any genomic DNA contamination, furthermore, genomic DNA contamination was

assayed by using an aliquot of whole-embryo uninjected RNA processed without

reverse transcriptase (labeled RT- in each experiment). Non-quantitative PCR on

the resulting cDNA was carried out for 35 cycles with an annealing temperature

of 55°C unless otherwise indicated.PCR products were separated on 2%

agarose gels and visualized with ethidium bromide. Ornithinedecarboxylase

(ODC) was used as a loading control for all RT-PCRs.

Primers:

odc: F: TTTGGTGCCACCCTTAAAAC R: CCCATGTCAAAGACACATCG xbra: F: AGGTTCAAGGAGCTCACCAA R: GAACCCATTCTCCATTCACG gsc: F: GTTGCACGTACAGACGCCTA R: TAAGGGAGCATTTGGTGAGG sox17b.1: F: GTTTATGGTGTGGGCAAAGG R: TCCTGCGTGGTCTGTACTTG mixer: F: TGTACCCAGACATCCACCAA R: CACTGGGGTAGCACATTCCT e2a: F: GTGGGTTCACCACAAGGAAT R: GTGCAGGAACAGTGACCTGA

heb: F: GGAGAGGGAAAAGGAGAGGA R: TATCAGTAAGCCCAGGATGC heb (splice junction spanning for MO) F: GGATGGCCGCTATAGGAAC R: TCCACTGAATTGACTGCTTCC

Quantitative real-time RT-PCR

Quantitative RT-PCR analysis was performed using SYBR green labeling system

(Bio-Rad) and the iCycler PCR machine and analysis software (Bio-Rad). RNA

isolation and cDNA preparation was carried out as for non-quantitative PCR, with

the exception that all mRNA amounts were quantified prior to reverse

transcription such that 1 µg of mRNA was used for each RT reaction. One µl of

the resulting cDNA was used in each 25 µl PCR reaction, together with 10 µM

primer mix and SYBR green reaction mix diluted to a final concentration of 1X.

The PCR reaction was as follows: 95°C for 5 min, 40 repeats with each repeat

consisting of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s. This was followed by

72°C for 7 min and a melt curve of 10 s cycles increasing by 0.5 degrees from

55°C to 95°C. After data collection, melt curves were analyzed visually to ensure

a single peak was present for each primer. Threshold amplification values (ct)

were assigned by the iCycler analysis software, and these were converted to

expression values following (Zhu et al., 2007), using either control-MO injected

embryo cDNA or ODC expression levels as a reference. Error bars represent

standard deviations.

Supplemental References

D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. 2005.

Efficient differentiation of human embryonic stem cells to definitive

endoderm. Nat Biotechnol 23:1534-1541.

Khokha MK, Chung C, Bustamante EL, Gaw LW, Trott KA, Yeh J, Lim N, Lin JC,

Taverner N, Amaya E, Papalopulu N, Smith JC, Zorn AM, Harland RM,

Grammer TC. 2002. Techniques and probes for the study of Xenopus

tropicalis development. Dev Dyn 225:499-510.

Kim SW, Yoon SJ, Chuong E, Oyolu C, Wills A, Gupta R, Baker JC. 2011.

Chromatin and transcriptional signatures for Nodal signaling during

endoderm formation in hESCs. Dev Biol, in press.

Lai JS, Herr W. 1992. Ethidium bromide provides a simple tool for identifying

genuine DNA-independent protein associations. Proc Natl Acad Sci U S A

89:6958-6962.

McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, Wenger AM,

Bejerano G. 2010. GREAT improves functional interpretation of cis-

regulatory regions. Nat Biotechnol 28:495-501.

Niewkoop PD, Faber J. 1967. Normal Table of Xenopus laevis. Amsterdam:

North Holland Publishing Company, reprinted 1994 Garland Publishing,

New York.

Pan Y, Ouyang Z, Wong WH, Baker JC. 2011. A New FACS Approach Isolates

hESC Derived Endoderm Using Transcription Factors. PLoS One

6:e17536.

Sive H. 2000. Xenopus Laevis Cold Spring Harbor Manual.

Zhu H, Gao W, Jiang H, Jin QH, Shi YF, Tsim KW, Zhang XJ. 2007. Regulation

of acetylcholinesterase expression by calcium signaling during calcium

ionophore A23187- and thapsigargin-induced apoptosis. Int J Biochem

Cell Biol 39:93-108.