Cell Reports, Volume 14

Supplemental Information

Tead and AP1 Coordinate Transcription and Motility

Xiangfan Liu, Huapeng Li, Mihir Rajurkar, Qi Li, Jennifer L. Cotton, JianhongOu, Lihua J. Zhu, Hira L. Goel, Arthur M. Mercurio, Joo-Seop Park, Roger J.Davis, and Junhao Mao

Table S5 (Related to Experimental Procedures). List of qPCR primers, shRNA and sgRNA sequences

qPCR primer 5'-3'

ABL2-F GGCAGAGGTATGGTCCTTGG

ABL2-R AAAGCTTCTGGACTACTGCCT

ANKRD1-F CGAGATAAGTTGCTCAGCACAG

ANKRD1-R GTTCAGTCTCACCGCATCATG

AJUBA-F ATATTATGCAGCAGCGGGGA

AJUBA-R CCCCATTGCTTGTAGGATCTTC

AMOTL2 AAAGCAGGTTAAAGGTGCTCCA

AMOTL2 CTCTGTGGGTGCTCTGTCTG

CDH2-F CCGGTTTCATTTGAGGGCAC

CDH2-R CCCATTGAGGGCATTGGGAT

CNN3-F GAAGCGAAGTGCGAGAGACC

CNN3-R TATAAGTTCGCAGAGGATGATGC

CTGF-F TGCCCTCGCGGCTTACCGACTG

CTGF-R TGCAGGAGGCGTTGTCATTGGTAAC

CYR61-F GAGTGGGTCTGTGACGAGGAT

CYR61-R GGTTGTATAGGATGCGAGGCT

DAAM1-F CAAGGTGCAGGAGTGGAGAG

DAAM1-R GTGTCAGCCTCTTCCTGGTC

DOCK1-F CCTAGACGCGGAGTTTCCTG

DOCK1-R ACCTCGGTACCACCCTTCAT

DOCK2-F GATGGGTGACCAGCACTACT

DOCK2-R TCCACAGCTGGAACTCAAAGT

DOCK3-F GGCTGAAAAAGGTCATTACTCCC

DOCK3-R CCGAGGAGTGCCCAAAGTTA

DOCK4-F ATGCTTGAGCAGGCACAGAT

DOCK4-R CTCACAGAAGCAGGTGCTGA

DOCK5-F GAAGCCGTCAAGATCAAGGGG

DOCK5-R GCACTTCTCTGCACTCTGACT

DOCK6-F AAACTGGATGCCCAGGTGAG

DOCK6-R CCGGGAGTCATTCGAGTCCT

DOCK7-F GCTCGGTACGTACACAATAGC

DOCK7-R ACAGAAACGGTCTGAACAACA

DOCK8-F ACCCTAGAAGCCACCGAACC

DOCK8-R ACTGAGGTAGTTGAAGAGAGGGG

DOCK9-F GCATATGCGCGAGCTTTCTT

DOCK9-R ATCATCCCAGCTGCTCATGC

GAPDH-F ATGGGGAAGGTGAAGGTCG

GAPDH-R GGGGTCATTGATGGCAACAATA

GPR126-F GAGCCAAGGTTGGTGCTTTG

GPR126-R GGCCAGTAGTAGCCTTTGGG

ITGB5-F TACCTGGAACAACGGTGGAG

ITGB5-R AAAAGATGCCGTGTCCCCAA

KIBRA-F GAGAAGCGGCAGATGGACC

KIBRA-R TCTGCAGAGAGAGCTGGGAT

LATS2-F CAGGATGCGACCAGGAGATG

LATS2-R GTAGGACGCAAACGAATCGC

MACF1-F GTCTTGTCGGAGTGAGCGAT

MACF1-R ATGTGCTTGCGGACCTTCAT

MKLN1-F CGCTACGGTGCTGACAAGAT

MKLN1-R AGAATCAAGTACTGGGGAGGA

NRP1-F ACGCAAGGCGAAGTCTTTTG

NRP1-R CAGTTGGCCTGGTCGTCAT

PARD3-F AGCAGAAATGCTGCCCTCAG

PARD3-R TCAAAGAGCAGTCGGCTGAA

PHLDB2-F CTCTGCTTGCTCACCAGACA

PHLDB2-R TTTCCCGTTCCCTGGATTCG

PTPN14-F TGCCCTCTTTCACACGGATG

PTPN14-R AGGTGTGCGTTTCCGAGTAG

TEAD1-F CCTCACAAGACGTCAAGCCT

TEAD1-R CTTGGTTGTGCCAATGGAGC

TEAD4-F AGTCAGGCACTGGACAAGC

TEAD4-R GCTGGAGACCTGCTTCCTG

TNS3-F TTAACCTCGAGTCTGCACGC

TNS3-R GGGAATCACCACCTTGGCTT

VGLL4-F GCTGTAATGGGAGGAAACCCA

VGLL4-R AGAGCAGCTTCGCGTTTTT

WTIP-F ATCACACGGTTTTTGCACCA

WTIP-R GCAACGACGACACAGTAGGT

YAP-F CCTCGTTTTGCCATGAACCAG

YAP-R GTTCTTGCTGTTTCAGCCGCAG

Primers of ChIP-qPCR 5’-3’

ANKRD1-F GGTGGTGATCACATCGCTCA

ANKRD1-R GGGGGTGTGATATGTAGGGC

AJUBA-F TCCAGTCCGCAGGTCTATCT

AJUBA-R CTCCACATCCAGGGCAGTTA

AMOTL2 CGGGCTAAGTGAATGCTTCC

AMOTL2 GGGGCGCCTATTATCACCTT

CTGF-F AGGCTTTTATACGCTCCGGG

CTGF-R TGAGTGTCAAGGGGTCAGGA

CYR61-F GCCAACCAGCATTCCTGAGA

CYR61-R GAGCCCGCCTTTTATACGGG

DOCK9-F GAGCAAAATAGCCAAGCGGC

DOCK9-R GCATGGAATCTGGAGTGGACA

KIBRA-F ATGTGGCTGAGCCGGTTTTA

KIBRA-R GGCAAGCAGGGTGAGGTAAT

LATS2-F GTCACTCTCGGCCGCATAC

LATS2-R GGCAGATAGCAGCCAGTCAA

PTPN14-F TTTTAAAAGGTGGGGGCCGA

PTPN14-R TCCTCGGAAGTCTAGGTGGG

TEAD1-F TCCTTTCCCAACCGTAAGACA

TEAD1-R CGCGGAAGTCTGCAAGTGTA

TEAD4-F CTTGGTAGGACTTTGCCGGA

TEAD4-R ATGGGTGCTGGGTCTCTTCT

VGLL4-F AGGAGGACCTCTGTACCTGC

VGLL4-R CCAGCTGCCCAGAGAGTCTA

WTIP-F CTGCGGAAATCCGAGCTCTTC

WTIP-R AGGAATGCGAGGAAGGAGACG

YAP-F GCCGGCTCACGGTATCTATTT

YAP-R AGGCCGAAAGAAGTGGAGAG

shRNA 5’-3’

Tead1/3/4 ATGATCA ACTTCATCCACAAG

YAP AAGCTTTGAGTTCTGACATCC

TAZ AGAGGTACTTCCTCAATCA

sgRNA for Crispr 5’-3’

Dock4 TACAGTTCAGATCCTGGAG

Dock9 GAAAGTCATCGTAAGGGAA

SRC1 GGTCGGATGAACTGTCCCCG

SRC2 GAAAAACGTAATCGTGAAC

SRC3 GAAAAACGGAGACGGGAGC

Tead2 GGAGGGTACCGGCGGCAGTG

Figure S1 (Related to Figure 1, 2)

A B C D E

F G H

Tead

4 IH

CTe

ad4

IHC

0%

50%

100%

Normal peripheralnerve tissue

Neuroblastoma

High expressionMedium expressionLow expression

I

(N=10)(N=50)

Pan-TEAD

TEAD1

TEAD4

GAPDH

J K

CTG

F m

RN

A le

vel (

%)

0

400

800

CaC

o2H

T29

FET

RKO

SW48

DLD

1H

CT1

16LS

174T

JunD:

DN-JunD:

AP1-

Luc

(%)

Anti-Flag

DBD

DBD

L

Flag tag

M

N

0

40

80

120 ControlDN-JunD

A. Homer de novo motif analysis of Tead4 ChIP-seq peaks in A549 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-818 -1.884e+03 37.85% 5.39% AP1

2

1e-557 -1.284e+03 41.04% 10.17% TEAD

3

1e-195 -4.507e+02 27.55% 10.47% FOXA1

4

1e-71 -1.656e+02 22.97% 12.55% CEBP

5

1e-49 -1.148e+02 11.66% 5.48% NFE2L1::MafG

B. Homer de novo motif analysis of Tead4 ChIP-seq peaks in HCT116 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-390 -8.995e+02 45.38% 5.10% AP1

2

1e-336 -7.743e+02 41.06% 4.87% TEAD

3

1e-33 -7.700e+01 11.09% 3.42% Tcf4

4

1e-29 -6.795e+01 17.95% 8.09% RUNX2

5

1e-27 -6.387e+01 2.70% 0.19% CTCF

C. Homer de novo motif analysis of Tead4 ChIP-seq peaks in SK-N-SH cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-1520 -3.501e+03 38.20% 5.81% AP1

2

1e-618 -1.425e+03 36.46% 12.53% TEAD

3

1e-250 -5.777e+02 40.17% 22.77% NFE2L1::MafG

4

1e-214 -4.950e+02 14.36% 4.91% RUNX

5

1e-114 -2.632e+02 21.80% 12.42% FEV

D. Homer de novo motif analysis of Tead4 ChIP-seq peaks in ECC1 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-1799 -4.143e+03 44.98% 12.94% TEAD

2

1e-665 -1.533e+03 13.48% 2.74% AP1

3

1e-210 -4.857e+02 33.84% 22.14% NFIC

4

1e-207 -4.778e+02 31.79% 20.47% SOX9

5

1e-203 -4.691e+02 25.49% 15.31% RELA

E. Homer de novo motif analysis of JunD ChIP-seq peaks in A549 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match

1

1e-2546 -5.864e+03 40.30% 3.11% AP1

2

1e-181 -4.173e+02 11.97% 4.12% FOXA1

3

1e-114 -2.635e+02 15.56% 7.82% Atf1(bZIP)

4

1e-99 -2.281e+02 26.08% 16.62% TEAD

5

1e-70 -1.628e+02 28.76% 20.31% POL003.1_GC-box

F. Homer de novo motif analysis of JunD ChIP-seq peaks in HCT116 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match

1

1e-3481 -8.016e+03 56.18% 3.63% AP1

2

1e-116 -2.688e+02 3.26% 0.41% NFE2L1::MafG

3

1e-101 -2.335e+02 16.32% 8.25% ERG(ETS)

4

1e-78 -1.804e+02 11.85% 5.78% Tcf

5

1e-75 -1.740e+02 40.76% 30.13% TEAD

G. Homer de novo motif analysis of JunD ChIP-seq peaks in SK-N-SH cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match

1

1e-4699 -1.082e+04 60.41% 7.18% AP1

2

1e-349 -8.054e+02 25.59% 11.90% TEAD

3

1e-203 -4.679e+02 17.30% 8.38% MafA

4

1e-170 -3.933e+02 9.87% 3.88% RUNX

5

1e-139 -3.222e+02 26.80% 17.31% NF1

H. Homer de novo motif analysis of Fra2 ChIP-seq peaks in A549 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-6520 -1.502e+04 48.80% 4.01% AP1

2

1e-364 -8.386e+02 14.38% 5.73% FOXA1

3

1e-184 -4.244e+02 23.80% 15.21% TEAD

4

1e-175 -4.036e+02 20.71% 12.85% cEBP-like_subclass

5

1e-78 -1.809e+02 2.71% 0.97% BORIS(Zf)

I. Homer de novo motif analysis of Fra1 ChIP-seq peaks in HCT116 cells

Rank Motif P-value log P-pvalue % of Targets % of Background Best Match/Details

1

1e-4409 -1.015e+04 61.68% 4.05% AP1

2

1e-130 -3.005e+02 4.86% 1.00% NFE2L1::MafG

3

1e-111 -2.571e+02 25.27% 15.31% ERG(ETS)

4

1e-54 -1.256e+02 13.97% 8.56% TEAD

5

1e-36 -8.374e+01 16.01% 11.18% NF1-halfsite(CTF)

J. Homer de novo motif analysis of Fra2 ChIP-seq peaks in SK-N-SH cells

Rank Motif P-value log P-pvalue % of

Targets % of

Background Best Match/Details

1

1e-5345 -1.231e+04 65.70% 5.21% AP1

2

1e-196 -4.515e+02 25.71% 13.97% TEAD

3

1e-97 -2.255e+02 32.55% 23.00% NF1-halfsite(CTF)

4

1e-90 -2.072e+02 5.81% 2.16% RUNX

5

1e-71 -1.636e+02 52.71% 43.49% Stat3(Stat)

Figure S2 (Related to Figure 1 and 2)

Figure S3 (Related to Figure 4)

inpu

tIP

JunD

JunD

IP:Tead4

Tead4

IgG

Tead4

─ ─ + : JNK-IN-8

CBA

0

50

100

150

Tead

-Luc

(%)

- JNK-IN-8 + JNK-IN-8

YAP/TA

ZDA

PI

ANK-

Luc

(%)

E

Anti-myc

D

0

600

1200

1800

Figure S4 (Related to Figure 5)

mR

NA

leve

l (%

)m

RN

A le

vel (

%)

A

B

0

50

100

150 ControlDN-JunDTeads KD/KODN-JunD + Teads KD/KO

0

40

80

120 ControlDN-JunDTead KD/KOTead KD/KO + DN-JunD

SUPPLEMENTAL FIGURE LEGENDS

Figure S1 (Related to Figure 1 and 2). Tead expression and inhibition of Tead and AP1

activity in cancer cells (A-I) Tead4 protein expression in human normal peripheral nerve tissue

and neuroblastoma tissue microarray samples. (A, B) Representative IHC images of Tead4

staining showing no or low expression of Tead4 proteins in human normal peripheral nerve

tissues (N=10). (C-H) Representative IHC images of Tead4 staining showing medium (C-E) or

high (F-H) expression of Tead4 proteins in human neuroblastoma tissue microarray samples

(N=50). (I) The percentile of low, medium or high level of Tead4 expression in normal

peripheral nerve and neuroblastoma tissues. (J) qPCR analysis of relative mRNA level of CTGF,

a known YAP/Tead target, in various colorectal cancer cell lines, indicating HCT116 as one of

the cell lines with high-level YAP/Tead activity. (K) Immunoblot analysis of overall Tead (Pan-

Tead), Tead1 and Tead4 proteins in HCT116 cells with or without Tead1-4 KD/KO expression.

(L) Diagram of JunD wild type and DN-JunD dominant repressor constructs. DBD: DNA

binding domain. (M) Immunoblot analysis of the expression of full length JunD and DN-JunD

proteins in transfected 293T cells, detected by an anti-flag tag antibody. (L) DN-JunD inhibits

JunD, cJun, Fos and Fra1-induced AP1-Luc activity in transfected 293T cells.

Figure S2 (Related to Figure 1 and 2). De novo motif analysis of Tead4, JunD and Fra1/2

ChIP-Seq peaks in A549, HCT116, SK-N-SH, and ECC1 cells. Enrichment of AP1 motif on

Tead4-occupied cis-regulatory regions in the genome of A549 (A), HCT116 (B), SK-N-SH (C)

and ECC1 (D) cells. Enrichment of Tead motif on JunD-occupied cis-regulatory regions in the

genome of A549 (E), HCT116 (F) and SK-N-SH (G) cells. Enrichment of Tead motif on Fra1/2-

occupied cis-regulatory regions in the genome of A549 (H), HCT116 (I) and SK-N-SH (J) cells.

Figure S3 (Related to Figure 4). JNK inhibition does not affect YAP activity and TEAD-

AP1 interaction in HCT116 cells. (A) Immunoflurescence staining of YAP/TAZ in HCT116

cells with and without JNK-IN-8 treatment. (B)Tead-dependent luciferase reporter activity in

HCT116 cells with and without JNK-IN-8 treatment. (C) JNK inhibition by JNK-IN-8 does not

affect endogenous JunD and Tead4 binding, measured by co-immunoprecipitation. (D)

Immunoblot analysis of the expression of the wild type cJun and mutated cJun4A proteins in

transfected 293T cells, detected by an anti-myc tag antibody. (E) Relative luciferase reporter

activity of ANK-Luc in HCT116 cells with cJun, cJun4A, or together with Fra1.

Figure S4 (Related to Figure 5). Transcription of the DOCK family factors and selected

Tead target genes in HCT116 cells with or without AP1 and Tead inhibition. (A) qPCR

analysis of transcription of the DOCK family guanine nucleotide exchange factors in HCT116

cells expressing Teads KD/KO, DN-JunD, or both. (B) qPCR analysis of transcription of the

selected Tead targets, including ABL2, CDH2, CNN3, DAAM1, GRP126, ITGB5, MACF1,

MKLN1, NRP1, PARD3, PHLDB2 and TNS3, in HCT116 cells expressing Teads KD/KO, DN-

JunD, or both.

Table S1 (Related to Figure 1). Tead4 ChIP-seq analysis in A549, HCT116, SK-N-SH, and

ECC1 genomes

Table S2 (Related to Figure 2). JunD ChIP-seq analysis in A549, HCT116, and SK-N-SH

genomes

Table S3 (Related to Figure 2). Fra1/2 ChIP-seq analysis in the A549, HCT116, and SK-N-

SH genomes

Table S4 (Related to Figure 5). List of the genes with Tead and AP1 co-occupied sites

identified in A549, HCT116 and SK-N-SH cancer cells.

Table S5 (Related to Experimental Procedures). List of qPCR primers, shRNA and sgRNA

sequences.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Cell culture, transfection, treatment and lentiviral infection. HCT116, SK-N-SH, ECC1, FET,

DLD1, LS174T, HEK293T cells were cultured in DMEM supplemented with 10% FBS. A549

cells were cultured in F-12K Medium supplemented with 10% FBS. HT29 cells were cultured in

McCoy 5A medium supplemented with 10% FBS. Caco2 and RKO cells were cultured in

EMEM medium supplemented with 20% FBS. SW48 cells were cultured in L15 medium

supplemented with 10% FBS. Transfection was performed using Lipofectamine 2000

(Invitrogen). Cells were treated with 1uM JNK Inhibitor JNK-IN-8 (EMD Millipore) or 20nM

Bufalin (Sigma) for 24 hours before subsequent analysis. For lentiviral infection, pLX or pGIPZ

based constructs were transfected along with the packing plasmids into growing HEK293T cells.

Viral supernatants were collected 48 hours after transfection, and target cells were infected in the

presence of polybrene and underwent selection with puromycin for 3-4 days. The lentiviral

expression vectors for Tead1, Tead2, SRC3, Fos, JunD, and Fra1 were purchased from DNASU

Plasmid Repository (https://dnasu.org). cJunWT-Myc and cJun4A-Myc were a gift from Axel

Behrens (Addgene plasmids #47443 and 47444). The cDNA fragments encoding human JunD,

DN-JunD, SRC3, SRC3-N, SRC3-M and SRC3-C were PCR cloned into a pLX or pGIPZ-based

lentiviral vector, and sgRNAs targeting human JunD, cJun, Tead2, SRC1/2/3, and Dock4/9 were

cloned into the pLentiCRISPR-V2 (a gift from Feng Zhang, Addgene plasmid # 52961). For

generation of pLKO-shTead1/3/4, shRNA against human Tead1/3/4 was cloned into pLKO.1-

blast (a gift from Keith Mostov, Addgene plasmid # 26655). The shRNA and sgRNA used for

targeting Teads, YAP/TAZ, SRC and Dock proteins were described in Supplemental Table S5.

Tead4, JunD, Fra1, Fra2, H3K4me1, H3K4me3, and H3K27ac ChIP-seq, de novo motif

discovery and functional clustering analyses. Tead4, JunD, Fra1, Fra2, H3K4me1, H3K4me3

and H3K27ac ChIP-seq data of A549, HCT116, SK-N-SH and ECC1 cells were downloaded

from the ENCODE project (http://genome.ucsc.edu/ENCODE/downloads.html). The antibodies

used in the ChIPseq analysis are as follows: Tead4 (SCBT, sc-101184), JunD (SCBT, sc74),

Fra1 (SCBT, sc183), Fra2 (SCBT, sc604), H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam,

ab8580) and H3K27ac (Abcam, ab4729) Peak calling and de novo motif analyses were done

using Homer (http://homer.salk.edu/homer/). To evaluate the statistical significance of Tead4,

JunD, Fra1/2 ChIP-seq peak overlaps, we sampled random matched genomic regions with the

same number of peaks and same peak length distribution as the identified peaks, and repeated the

process 100 times to obtain the empirical null distribution of the number of overlaps between

identified sites and random regions. The Tead4, JunD, H3K4me3 and H3K27ac tracks of the

ANKRD1, Dock9 and Tead4 loci were generated by Bioconductor package trackViewer

developed by J Ou and LJ Zhu. Functional annotation and clustering analyses of the list of the

genes carrying Tead4 and/or AP1 peaks were done using Database for Annotation, Visualization

and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/).

Immunohistochemistry, immunofluorescence and immunoblotting. Immunohistochemistry

was performed on 5μm sections of formalin fixed and paraffin embedded tumors. Following

rehydration, antigen retrieval was performed at 100 °C for 0.5-2 hours with Tris-EDTA buffer.

Endogenous peroxidase activity was inhibited by incubating with 3% peroxide solution for 20

minutes. Sections were then blocked in a blocking buffer containing 5% BSA and 0.1% Triton

X-100, and incubated overnight at 4℃ in primary antibodies against Tead4 (Abcam, ab97460,

1:100) and JunD (Cell signaling, 5000S, 1:50). Signal detection was accomplished with

biotinylated secondary antibodies in the Vectastain ABC kit (Vector Labs).

For immunofluorescence, cells were fixed by 4% paraformaldehyde for 5 minutes, washed with

PBS and blocking buffer (PBS with 5% BSA and 0.1% Triton X-100), and then incubated

overnight at 4℃ in primary antibodies against JunD (Abcam, ab134067), Fra1 (SCBT, SC183),

YAP/TAZ (Cell signaling, 8418s), and SRC1 (Cell signaling, 2191). Alexa-Fluor fluorescent

conjugated secondary antibodies (Invitrogen) were used for detection.

Immunoblotting was performed using standard protocol. The primary antibodies used were: Flag

(Sigma, F3165), Myc (Developmental Studies Hybridoma Bank), V5(Thermo Fisher, MA5-

15253),YAP (Cell signaling, 4912), p-YAP (Cell signaling, 4911), TAZ (BD, 560235)), Tead4

(Abcam, ab97460), pan-Tead (Cell signaling, 13295s), JunD (Abcam, ab134067), cJun (Cell

signaling, 9165), p-cJun (Cell signaling, 9164), Fra1 (SCBT, sc183), Fra2 (SCBT, sc604),

ANKRD1 (Abnova, H00027063-B02P), Tead1 (Abcam, ab133533), Tead2 (Cell Signaling, 887),

Dock4 (Bethyl, A302-263A-T), Dock9 (Bethyl, A300-530A-T), RhoA (Cytoskeleton),

RAC1(Cytoskeleton), SRC1 (Cell signaling, 2191), SRC2 (Bethyl, A300-346A-T), SRC3 (Cell

signaling, 2126) and GAPDH (Santa Cruz, 5147). HRP conjugated secondary antibodies used for

detection were obtained from Jackson Laboratories.

Soft agar colony formation, cell migration and invasion, and small G protein activation. For anchorage-independent soft-agar colony formation assay, cells were seeded at a density of

6000 cells/well in a 6-well plate of 0.3% agarose in DMEM media containing 10% FBS.

Colonies from 12 fields of view were counted 14 days later. Assays were conducted in triplicates,

and standard deviation was used to calculate error bars. For cell scratch assay, the cell monolayer

was scratched in a straight line with a p200 pipet tip, and images were taken at 0 or 48 hours

later. For transwell migration assays, 5X104 cells were plated in the top chamber with the non-

coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). For Matrigel invasion

assays, 2X105 cells were plated in the top chamber with Matrigel-coated membrane (24-well

insert; pore size, 8 mm; BD Biosciences). In both assays, cells were plated in medium without

serum or growth factors, and medium supplemented with serum was used as a chemoattractant in

the lower chamber. The cells were incubated for 24h and cells that did not migrate or invade

through the pores were removed by a cotton swab. Cells on the lower surface of the membrane

were stained with the Diff-Quick Staining Set (Dade) and counted. RhoA/RAC1/CDC42 G-

LISA activation assays (Cytoskeleton) were performed according to the manufacturer’s protocol.

Immunoprecipitation and chromatin immunoprecipitation. HCT116 cells or transfected

HEK293T cells were lysed using lysis buffer (50 mM Tris HCl, pH 8.0, 170 mM NaCl, 0.5%

Nonidet P-40, 50 mM NaF and 1 mM PMSF). Cell lysates were centrifuged at 14,000 × g for 10

minutes, and supernatants were used for immunoprecipitation. After preclearing for 1 hour with

protein A/G-Sepharose (Invitrogen), supernatants were incubated at 4℃ overnight with

antibodies and protein A/G-Sepharose beads. Immunoprecipitates were washed five times with

lysis buffer, and proteins were eluted with SDS sampling buffer, and analyzed by

immunoblotting using the antibodies as indicated. Trueblot secondary antibodies (Rockland)

were used for immunoblot detection.

Chromatin immunoprecipitation ChIP assays were performed according to the manufacturer’s

instructions (Active Motif, CA). Briefly, 2×107 cells were fixed with 1% formaldehyde, washed

with cold PBS and lysed in lysis buffer. After sonication, protein-DNA complexes were

incubated with antibody-coupled protein G beads at 4℃ overnight. After elution in 1%

SDS/0.1M NaHCO3 and reverse cross-link at 65℃, DNA was purified by phenol/chloroform

extraction and ethanol precipitation, and subjected to PCR or qPCR analysis. The primers used

for real-time PCR of cis-regulatory elements were listed in Table S5. In the Re-ChIP

experiments, complexes were eluted by incubation for 30 minutes at 37℃ in 10mM DTT. After

centrifugation, the supernatant was diluted 20 times with Re-ChIP buffer (1% Triton X-100,

2mM EDTA, 150mM NaCl, 20Mm Tris-HCl, pH 8.1) for further ChIP-qPCR analysis. The

primers used for ChIP-qPCR analysis were described in Supplemental Table S5.

Quantitative Real-Time PCR. RNA of cultured cells was isolated using Trizol reagent

(Invitrogen), followed by reverse-transcription using SuperscriptII Reverse Transcriptase

(Invitrogen). Quantitative real-time PCR was performed using Sybr Mastermix (Kapa

Bioscience). The primers used for real-time PCR were described in Supplemental Table S5. All

qPCR experiments were conducted in biological triplicates, error bars represent mean ±

standard deviation, and Student’s t-test was used to generate p-values.

Luciferase reporter analysis. Tead-Luc (8xGTIIC-luciferase) was a gift from Stefano Piccolo

(Addgene plasmid # 34615). AP1-Luc (3xAP-1 in pGL3-basic) was a gift from Alexander Dent

(Addgene plasmid # 40342). The ANK-Luc, Dock9-Luc, Tead4-Luc, and ANK-Luc mutated

constructs were generated by PCR and subcloning into pGL3 based luciferase vectors (Promega).

Cells were transfected with different reporter constructs together with a Renila luciferase

expression plasmid for 48 hours. Luciferase activity was measured as using the Dual-Luciferase

reporter assay system (Promega) according to the manufacturer’s protocol. All luciferase

reporter experiments were conducted in biological triplicates, error bars represent mean ±

standard deviation, and Student’s t-test was used to generate p-values.