R E S O U R C E
Genome-wide association between YAP/TAZ/TEAD
and AP-1 at enhancers drives oncogenic growth
Francesca Zanconato
1
, Mattia Forcato
2
, Giusy Battilana
1
, Luca Azzolin
1
, Erika Quaranta
1
, Beatrice Bod ega
3
,
Antonio Rosato
4,5
, Silvio Bicciato
2
, Michelangelo Cordenonsi
1,6
and Stefano Piccolo
1,6
YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as
transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ
transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA,
co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements
harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes
directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that
contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and
severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice.
This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.
YAP/TAZ (refs 1,2) are potent inducers of cell proliferation and
are important drivers of tumorigenesis in a number of contexts
3–6
.
Yet, the mechanisms underpinning this activity remain enigmatic.
Only a handful of direct targets have been described in mammalian
cells, leaving largely undefine d what are the immediate downstream
effectors by which YAP/TAZ exert their biological effec ts
3
. More over,
lack of systematic studies results in just a scattered understanding
of the transcriptional partners by which nuclear YAP/TAZ control
transcription on a genome-wide scale. Also unknown is whether and
how, after binding to DNA, YAP/TAZ achieve combinatorial control of
gene expression, for example through cooperation with other nuclear
oncogenes during YAP/TAZ-driven oncogenic growth.
RESULTS
A genomic map of YAP/TAZ recruitment to chromatin
To elucidate how YAP/TAZ regulate gene expression in tumour cells
we performed chromatin immunoprecipitation assays with YAP and
TAZ antibodies followed by next-generation sequencing (ChIP-seq)
in MDA-MB-231 breast c ancer cells, bearing genetic inactivation
of the Hippo pathway (NF2 null)
7
. A total of 7,107 peaks were
identified by both antibodies (Fig. 1a). YAP/TAZ-bound regions
included the promoters of previously established YAP/TAZ direct
targets (CTGF, CYR61, ANKRD1, AXL, AMOTL2, AJUBA and WTIP,
Supplementary Fig. 1b–d).
YAP/TAZ do not carry a DNA-binding domain, and thus
can contact the DNA only indirectly, through transcription factor
partners. So far, a number of these partners have been described,
including TEAD1-4, RUNX, p73, KLF4, TBX5 and others
3,8–11
. Motif
analyses at YAP/TAZ peaks revealed that the main platforms by which
YAP/TAZ interact with DNA are TEAD proteins: their consensus
sequence was present in at least 75% of YAP/TAZ peaks, typically
at the summit of YAP/TAZ peaks (Supplementary Fig. 1e,f). TEAD
factors have been repeatedly ass ociated as mediators of YAP/TAZ
transcriptional responses
3,9
; surprisingly no motifs for the other
proposed DNA-binding platforms of YAP/TAZ were significantly
enriched (with the exception of RUNX, found in a minority of peaks;
see Supplementary Table 1).
Follow ing these results, we performed a ChIP-seq experiment
for endogenous TEAD4 and found that 78% (5,522) of YAP/TAZ
peaks overlapped with TEAD4 peaks (Fig. 1b and Supplementary
Table 2); furthermore, the summits of TEAD4 peaks coincide with the
summit of the corresponding YAP/TAZ peaks (Fig. 1c), indicating that
TEAD factors are indeed the main driver for YAP/TAZ recruitment
to chromat in. In support of this notion, the signal of TEAD4 peaks
1
Department of Molecular Medicine, University of Padua School of Medicine, viale Colombo 3, 35126 Padua, Italy.
2
Center for Genome Research, Department of Life
Sciences, University of Modena and Reggio Emilia, via G. Campi 287, 41100 Modena, Italy.
3
Genome Biology Unit, Istituto Nazionale di Genetica Molecolare (INGM)
‘Romeo and Enrica Invernizzi’, via Francesco Sforza 35, Milan 20126, Italy.
4
Department of Surgery, Oncology and Gastroenterology, University of Padua School of
Medicine, Via Gattamelata 64, 35126 Padua, Italy.
5
Istituto Oncologico Veneto IOV-IRCCS, Via Gattamelata 64, 35126 Padua, Italy.
6
Correspondence should be addressed to M.C. or S.P. (e-mail: michelangelo.cordenonsi@unipd.it or piccolo@bio.unipd.it)
Received 20 March 2015; accepted 2 July 2015; published online 10 August 2015; DOI: 10.1038/ncb3216
1218 NATURE CELL BIOLOGY VOLUME 17 | NUMBER 9 | SEPTEMBER 2015
© 2015 Macmillan Publishers Limited. All rights reserved
R E S O U R C E
0
5
10
15
20
0
10
20
30
40
0 10203040
TEAD4 signal
0
10
20
30
40
0 10203040
TEAD4 signal
YAP signal
YAP/TAZ/TEAD peaks
TAZ signal
r
2
= 0.43 r
2
= 0.46
Anti-TAZ
9,798
Anti-YAP
7,709
YAP/TAZ
7,107 peaks
n of peaks
a
n of peaks
TEAD
8,406
YAP/TAZ
7,107
YAP/TAZ
TEAD
5,522 peaks
0
0.5
1.0
1.5
2.0
–250 0 250
Position of TEAD4
peak summit
Distance to the summit
of TAZ peaks (bp)
Peak density (×10
2
)
CO siRNA TEAD siRNA
Relative DNA binding
Negative
control
ChIP: YAP
ANKR D1
AXL
Promoters
Active enhancers
Inactive enhancers
Not classified
YAP/TAZ/TEAD peaks
2.6%
5.3%
3.6%
88.5%
0
10
20
30
40
50
60
Percentage of peaks
YAP/TAZ peaks
associated with
regulated genes
Distance to TSS
(kb)
H3K4me1
YAP
TAZ
H3K4me3
H3K27ac
Promoter
Active
enhancer
Inactive
enhancer
Not
assigned
TEAD4
TAZ
YAP
–1 kb +1 kb
–1 kb 0 +1 kb
Summit
of peak
me1 me3
H3K4
Promoters
Enhancers
TEAD4
0
0.1
0.2
0.3
0.4
0.5
0.6
YAP
TAZ H3K4me1
Normalized
read density
Distance to peak summit
TEAD4
0
20
40
60
80
100
YAP
peaks
TAZ
peaks
TEAD4
peaks
YAP/TAZ/
TEAD
Distance to TSS
%
<1 kb
1–10 kb
10–100 kb
>100 kb
<1
1–10
10–100
>100
d
hi kj
fg
bc
e
Figure 1 Genome-wide co-localization of YAP, TAZ and TEAD on enhancers.
(a) Overlap of peaks identified with YAP and TAZ antibodies. See
Supplementary Fig. 1a for specificity controls of the antibodies, and
Supplementary Table 1 for the results of de novo motif finding in YAP/TAZ
peaks. (b) Overlap of YAP/TAZ peaks and TEAD4 peaks. See Supplementary
Fig. 1g for specificity controls of TEAD4 antibody, and Supplementary Fig. 1h
for ChIP-seq profiles at positive control loci. (c) Position of TEAD4 peak
summits relative to the summits of the overlapping YAP/TAZ peaks, in
a 500 bp window surrounding the summit of YAP/TAZ peaks. (d) Linear
correlation between the signal of YAP or TAZ and TEAD4 peaks in the
5,522 shared binding sites. r
2
is the coefficients of determination of the
two correlations. (e) ChIP-qPCR showing YAP binding to the indicated
sites in MDA-MB-231 cells transfected with control (CO siRNA) or TEAD
siRNAs (TEAD siRNA A). Relative DNA binding was calculated as fraction
of input and normalized to IgG (IgG bars are omitted); data from 2
biological replicates (individual data points and their mean) from one
representative experiment are shown. (f) Absolute distance of YAP peaks
(n= 7,709), TAZ peaks (n = 9,798), TEAD4 peaks (n = 8,406) or overlapping
YAP/TAZ/TEAD peaks (n = 5,522) to the nearest TSS. (g,h) Association of
YAP/TAZ/TEAD peaks to promoters and enhancers according to ChIP-seq
data for histone modifications. (g) Scheme illustrating peak classification.
(h) Fraction of YAP/TAZ/TEAD peaks associated with each category. See
Supplementary Fig. 1j for validation of the enhancer/promoter status of a set
of YAP/TAZ/TEAD-bound regions. (i) Heatmap representing YAP/TAZ/TEAD-
binding sites located on promoters (top) and enhancers (bottom). YAP,
TAZ and TEAD4 peaks are ranked from the strongest to weakest signal
in TAZ ChIP, in a window of ±1 kb centred on the summit of TAZ
peaks. H3K4me1 and H3K4me3 signal in the corresponding genomic
regions is shown on the right. (j) Bimodal distribution of H3K4me1
signal around the summit of YAP/TAZ and TEAD4 peaks. (k) Distance
between YAP/TAZ/TEAD-binding sites and the TSS of the direct target
genes they are associated to. Overall, 635 peaks were associated to 379
genes positively regulated by YAP/TAZ. See Methods for reproducibility
of experiments.
is positively correlated with that one of YAP/TAZ peaks (Fig. 1d),
and binding of YAP to chromatin is strongly affected by combined
knockdown of TEAD1/2/3/4 as assessed by ChIP-qPCR (Fig. 1e).
Global association of YAP/TAZ/TEAD to enhancer elements
We analysed the distribution of YAP-, TAZ- or TEAD-binding sites
relative to genes annotated in the human genome, and found that
only a minute fraction of peaks mapped close (±1 kilobase (kb))
to transcription start sites (TSSs) whereas most peaks were located
farther than 10 kb f rom the closest TSS (Fig. 1f). Analyses of publicly
available TEAD ChIP-seq data revealed that this pattern is conserved
in several cancer cell t ypes (Supplementary Fig. 1i).
Owing to t heir remote location, we questioned whether most
YAP/TAZ/TEAD common pe aks are located in enhancers. Enhancers
NATURE CELL BIOLOGY VOLUME 17 | NUMBER 9 | SEPTEMBER 2015 1219
© 2015 Macmillan Publishers Limited. All rights reserved
R E S O U R C E
Biological function of YAP/TAZ
direct positive targets
Cell proliferation
RNA metabolism
Other GO terms
35.6%
13.5%
50.9%
CO siRNA
YT siRNA1
YT siRNA2
0
1
qRT–PCR
TEAD siRNA A
TEAD siRNA B
a
25 kb
Interaction with
TOP2A promoter
38,670,000 38,720,000
Position in chromosome 17
38,770,000
1.0
0.5
0
prey
YTT-binding sites
TOP2A
anchor
IGFBP4 TNS4 CCR7
40 kb
YTT-binding sites
TOP2A enh2/3
Tested
interactions
prey
20 kb
Interaction with
MYC promoter
YTT-binding site
128,210,000 128,260,000
Position in chromosome 8
100 kb
TAZ
TEAD4
YTT-binding site
MYC enh1
MYCPOU5F1B
RefSeq
genes
Tested
interactions
TAZ
TEAD4
RefSeq
genes
1.0
0.5
0
0
0.5
1.0
Relative levels
H3K27ac
MYC
enh1
MYC
enh4
TOP2A
enh1
CO siRNA YT siRNA
CCNA2
CDC25A
CDC6
CDCA5
CDCA8
CENPF
CENPV
EDN1
ETS1
ETS2
FOSL1
GADD45B
GINS1
KIF14
KIF20B
KIF23
KNTC1
MCM3
MCM7
MRE11A
MYBL1
MYC
POLA2
POLE3
POLH
PSMC3IP
PSMG1
RAD18
RRM2
RUVBL2
SGOL1
SMC3
SUV39H2
TIMELESS
TK1
TOP2A
TUBB
ZWILCH
cd
b
e
anchor
Figure 2 YAP/TAZ/TEAD transcriptional program. (a) Biological functions
associated to YAP/TAZ direct positive targets, identified by GO terms.
(b) YAP/TAZ or TEAD depletion impairs the expression of YAP/TAZ/TEAD
direct target genes involved in cell proliferation, as evaluated by qRT–PCR
(CO siRNA, control siRNA; YT siRNA, YAP/TAZ siRNA). For a subset of
genes, the downregulation of the corresponding proteins was also verified
by western blot (Supplementary Fig. 2b). See Supplementary Fig. 2a for
validation of TEAD siRNAs. (c,d) Validation of the long-range interaction
between YAP/TAZ-occupied enhancers and the promoters of MYC (c) and
TOP2A (d) by DNA looping, using 3C assays in MDA-MB-231 cells. TAZ
and TEAD4 ChIP-seq profiles show the position of YAP/TAZ/TEAD-binding
sites upstream of MYC or TOP2A genes (here named ‘MYC enhancer 1’,
‘TOP2A enhancer2’ ‘TOP2A enhancer3’), whereas no YAP/TAZ/TEAD-binding
sites were detected close to their TSS. The chart shows the frequency of
interaction (measured as crosslinking frequency) between MYC or TOP2A
promoter (‘anchor’) and the indicated sites surrounding YAP/TAZ/TEAD (YTT)
peaks (green lines). Interaction frequency is higher close to YAP/TAZ peak.
Data points are mean + s.e.m. from n = 3 biological replicates. See also
Supplementary Fig. 2d,e for the additional interactions between MYC and
TOP2A promoters and a different set of YAP/TAZ/TEAD-bound enhancers.
(e) ChIP-qPCR comparing the levels of H3K27ac (normalized to total
H3 levels) in MDA-MB-231 cells transfected with control (CO siRNA) or
combined YAP/TAZ siRNAs (YT siRNA, a mix of YT siRNA 1 + YT siRNA 2).
Data from 2 biological replicates (individual data points and their mean) from
one representative experiment are shown. See Methods for reproducibility
of experiments.
can be distinguished from promoters by their epigenetic features, that
is, relative enrichment of histone H3 monomethylation (H3K4me1)
on Lys 4 at enhancers, and trimethylation (H3K4me3) at promoters
12
.
ChIP-seq data for these epigenetic marks in MDA-MB-231 cells were
recently reported
13
, allowing us to compare this map of promoters
and en hancers with the location of YAP/TAZ/TEAD4 peaks (Fig. 1g).
Notably, only a very small fraction (3.6%) of YAP/TAZ/TEAD4 peaks
are located on promoters; instead, 91% of peaks are located in
enhancer regions (Fig. 1h,i). Furthermore, most of these enhancers
are in an active state as revealed by H3K27 acetylation and reduced
nucleosome occupancy at the peak centre, also resulting in a bimodal
distribution of H3K4me1 around the peak summit (Fig. 1h–j and
Supplementary Fig. 1k).
A YAP/TAZ-regulated transcriptional program driving cell
proliferation
We then sought to link YAP/TAZ/TEAD4 peaks to corresponding
target genes (Supplementary Fig. 1l). All of the peaks located in
promoter regions (201) were assigned to the nearest genes. However,
application of this proximity criterion to the distant enhancers bound
by YAP/TAZ was questionable, as enhancers can regulate target genes
over long distances, often skipping intervening genes. Instead, the
specificity of enhancer–promoter associations is dictated by the three-
dimensional higher-order chromatin struc ture, whereby enhancers
interact with their target promoters through chromat in looping
12
.
Importantly, a recently reported high-resolution map of chromatin
interactions (Hi-C) has been shown to predict bona fide enhancer–
promoter pairs with great accuracy
14
. Notably, most of these long-
range chromatin interactions are conserved across cell types
15
. By
using the map of enhancer–promoter pairs discovered in ref. 14, we
could associate more than half of YAP/YAZ/TEAD4-b ound enhancers
to a set of 2,957 candidate target genes; considering also the genes
with peaks in their promoters, the list extends to 3,089 genes. Of these
candidates, 379 are in fact expressed in MDA-MB-231 cells, and in a
YAP/TAZ-dependent manner, as determined by Affymetrix profiling
of control and YAP/TAZ-depleted cells (using two independent
1220 NATURE CELL BIOLOGY VOLUME 17 | NUMBER 9 | SEPTEMBER 2015
© 2015 Macmillan Publishers Limited. All rights reserved
R E S O U R C E
combinations of siRNAs; Supplementary Table 3). Crucially, most
(88.6%) of these bona fide YAP/TAZ direct targets are controlled only
from distal enhancers, mostly located farther than 100,000 base pairs
(bp) from the T SS (Fig. 1k); we further note that these genes could not
have been identified by assigning the peak to the closest gene.
To identify the main biological processes regulated by YAP/TAZ,
we performed Gene Ontology (GO) analyses on the list of YAP/TAZ
direct targets. A large fraction of positive targets (135) are linked to
processes related to cell cycle progression (Fig. 2a and Supplement ary
Tables 4 and 5); YAP/TAZ/TEAD-binding sites are loc ated exclusively
on distal enhancers for 115 of t hese genes, and both on promoters and
enhancers for other 11 genes. Other positive targets (13.5% of the total)
are connected to RNA metab olism and RNA transport.
The YAP/TAZ/TEAD cell-proliferation program comprises
essential factors involved in replication licensing, DNA synthesis and
repair (for example, CDC6, GINS1, MCM3, MCM7, POLA2, POLE3,
TOP2A and RAD18), transcriptional regulators of the cell cycle
(for example, ETS1, MYC and MYBL1), cyclins and their activators
(CCNA2 and CDC25A), and factors required for completion of
mitosis (for example, CENPF, CDCA5 and KIF23). We selected
about 40 of these genes (containing representative genes belonging
to each of the above-mentioned categories) and confirmed by
quantitative PCR with reverse transcription (qRT–PCR) that their
expression depends on YAP/TAZ as well as on TEAD1-4 (Fig. 2b).
All of these new YAP/TAZ/TEAD-regulated genes were associated
to YAP/TAZ/TEAD peaks located on enhancers (as exemplified in
Supplementary Fig. 2c). By chromatin conformation capture (3C)
analysis we confirmed that the interaction of YAP/TAZ/TEAD-bound
enhancers with MYC or TOP2A promoters occurs through chromatin
looping in MDA-MB-231 cells (Fig. 2c,d and Supplementary
Fig. 2d,e), validating the procedure here used to associate distant
enhancers to target genes by using Hi-C data. Remarkably, YAP/TAZ
are required for the activity of these enhancers, as acetylation of
H3K27 in these regions drops in YAP/TAZ-depleted cells (Fig. 2e).
We next aimed to determine the biological validity of these
findings. We found that YAP/TAZ-depleted cells stop proliferating and
accumulate in the G
1
phase (Fig. 3a,b). These effects are phenocopied
by TEAD depletion (Supplementary Fig. 3b,c); moreover, the growth
of YAP/TAZ-depleted cells can be fully rescued by reintroduction
of wild-type YAP or TAZ, but not by their TEAD-binding-deficient
mutants (Fig. 3c and Supplementary Fig. 3d). Together, these results
underline the relevance of TEAD as the master determinant of
YAP/TAZ-driven proliferation
9
.
To investigate the newly identified YAP/TAZ direct targets, we
focused on MYC, given its established prominence as a regulator of cell
proliferation. As shown in Supplementary Fig. 3e–g, MYC knockdown
caused a minor, but significant reduction of cell proliferation and a
substantial increase of cells in the G
1
phase, in part phenocopying
the requirement of YAP/TAZ. Overexpression of MYC in YAP/TAZ-
depleted cells triggered a significant, but only a partial rescue of
cell proliferation (Fig. 3d). This indicates that MYC represents an
important functional effector of YAP/TAZ in this context; however,
MYC alone cannot fully recapitulate the biological effectiveness
of YAP/TAZ.
We next sought to determine whether genes identified as
YAP/TAZ/TEAD targets in MDA-MB-231 cells are exploited in
human breast cancers. YAP/TAZ are required and sufficient to
confer malignant traits to more benign tumour cells
16–18
. In line,
elevated levels of YAP/TAZ in human breast cancer specimens identify
aggressive tumours (defined as high histopathological grade, or ‘G3’),
and those with worse prognosis
16
. Direct targets of YAP/TAZ/TEAD
should thus similarly earmark aggressive tumours and be prognostic.
To test this idea, we used a data set of >3,600 clinically annotated
and transcr iptionally profiled breast cancer samples (Supplementary
Table 8) and confirmed that the signature enlisting the validated
YAP/TAZ/TEAD direct targets (see Fig. 2b) was differentially
expressed in G3 and G
1
tumours, and identified tumours with
poor prognosis as determined by Kaplan–Meier analyses (Fig. 3e,f).
Moreover, the signature of direct targets statistically correlates with
the expression of independent YAP/TAZ signatures, and with the
levels of TAZ messenger RNA, which is amplified in a subset of
breast tumours
16,19
(Supplementary Fig. 3i). Thus, the expression of
direct YAP/TAZ/TEAD target genes here identified correlates with
YAP/TAZ activation in human tumours.
Genomic co-occupancy of AP-1 and YAP/TAZ/TEAD
De novo motif analyses in YAP/TAZ/TEAD peaks revealed that, after
TEAD consensus, the second most frequent motif corresponded to
the consensus for AP-1 transcription factors (Supplementary Table 1).
AP-1 are dimers of JUN (JUN, JUNB, JUND) and FOS (FOS, FOSB,
FOSL1 and FOSL2) families of leucine-zipper proteins
20
. Many of
these factors are archetypal oncogenes involved in the control of
cellular growth and neoplastic transformation. Most YAP/TAZ/TEAD
peaks (70%) contained both a TEAD and an AP-1 motif, with a median
distance of about 60 bp.
We next verified that AP-1 factors are indeed recruited to the same
genomic regions bound by YAP/TAZ/TEAD4. For this, we considered
JUN as a surrogate mark for bound AP-1 dimers, because it is a
common component of JUN/FOS and JUN/JUN dimers
20
. By ChIP-
seq we identified >24,000 JUN-binding sites. JUN was present in 78%
of YAP/TAZ/TEAD-binding sites (4,306/5,522; Fig. 4a,b), and 93% of
these shared binding sites are located on active enhancers. This is in
line with the notion that composite TEAD and AP-1 motifs dominate
the YAP/TAZ cistrome.
JUN peaks were detected on the regulatory regions of well-
established YAP/TAZ/TEAD target genes (CTGF and ANKRD1—
Supplementary Fig. 4c), and on the enhancers of 85% of the new
targets defining the YAP/TAZ/TEAD cell-proliferation program (as in
Fig. 4c). We indeed re-validated by ChIP-qPCR the presence of both
JUN and FOSL1 in all tested binding sites (Supplementary Fig. 4d,e).
By considering ChIP-seq data from the ENCODE project
21
, TEAD4
and AP-1 peaks largely overlap in all examined tumour cell lines
(Supplementary Fig. 4f,g), indicating that co-occupancy of TEAD
and AP-1 transcription factors on the same regions is a widespread
phenomenon. To test the possibility that AP-1 proteins and YAP/TAZ
can simultaneously co-occupy chromatin, we carried out a sequential
ChIP for YAP followed by anti-JUN reChIP at selected loci. The results
indicated that both factors co-occupy the same cis-regulatory elements
at the same time (Fig. 4d).
Given their vicinity on DNA, we tested whether YAP/TAZ, TEAD
and AP-1 proteins could physically interact. By in situ proximity
ligation assay
22
, we found YAP and TEAD1 in close proximity with
NATURE CELL BIOLOGY VOLUME 17 | NUMBER 9 | SEPTEMBER 2015 1221
© 2015 Macmillan Publishers Limited. All rights reserved
R E S O U R C E
0
0.5
1.0
CO siRNA
CO siRNA + doxy
YT siRNA1
YT siRNA1 + doxy
Normalized absorbance
MDA TetON MYC
12345
Day
0
0.5
1.0
CO siRNA
YT siRNA1
YT siRNA2
Normalized absorbance
Day
Cell growth
1
234
a
–4
–2
0
2
4
Grade
1
Grade
2
Grade
3
Signature expression
Human breast tumours
0
0.2
0.4
0.6
0.8
1.0
Normalized absorbance
Day 1 Day 5
Empty
vector
Empty
vector
TAZ
wt
TAZ
S51A
CO siRNA YT siRNA1
Cell growth
024681012
0
20
40
60
80
100
Time (yr)
P value < 0.0001
Percentage of survival
Low
High
Metastasis-free survival
0
20
40
60
80
100
G
1
SG
2
/M
Cell cycle phases
YT siRNA2
YT siRNA1
CO siRNA
Percentage of cells
de
b
f
c
Figure 3 Control of cell proliferation by YAP/TAZ/TEAD. (a) Growth curve
of MDA-MB-231 cells transfected with control siRNA (CO siRNA) or two
different combinations of siRNAs targeting YAP and TAZ (YT siRNA). Data
are mean + s.d. of n = 8 biological replicates. Individual depletion of YAP or
TAZ has no effect on cell growth (Supplementary Fig. 3a). (b) Percentage of
MDA-MB-231 cells in G
1
, S and G
2
/M phases of cell cycle, as determined
by flow-cytometric analysis of DNA content. Cells were transfected with
control (CO siRNA) or YAP/TAZ siRNAs (YT siRNA) 48 h before fixation. Data
are mean + s.d. of n = 3 biological replicates. (c) Sustained expression of
TAZ, but not of TEAD-binding-deficient TAZS51A, rescues cell proliferation
in YAP/TAZ-depleted cells. Empty-vector-, wild-type TAZ- (wt) or TAZS51A-
transduced MDA-MB-231 cells were transfected with control (CO siRNA)
or YAP/TAZ (YT siRNA) siRNAs, as indicated. Proliferation was evaluated
as in f. Data are mean + s.d. of n = 8 biological replicates. (d) MDA-
MB-231 cells were transduced with lentiviral vectors encoding rtTA and
doxycycline-inducible MYC (MDA TetON MYC) and transfected with control
or YAP/TAZ siRNAs. Where indicated, MYC expression was induced with
0.1 µg ml
−1
doxycycline at the time of transfection. Cell growth was evaluated
as in f. Data are mean + s.d. of n = 8 biological replicates. A control
experiment with doxycycline-inducible EGFP is shown in Supplementary
Fig. 3h. (e) Average gene expression values of validated YAP/TAZ/TEAD direct
target genes (listed in Fig. 2b) in invasive breast cancer samples, classified
according to their histological grade. Individual data points (n = 3,661
patient samples) and the mean value (black line) of each group are
shown. (f) Kaplan–Meier graph representing the probability of cumulative
metastasis-free survival in breast cancer patients stratified according to
the expression of validated YAP/TAZ/TEAD direct target gene signature.
High expression of the signature is associated with shorter metastasis-
free survival (log-rank P value < 0.0001). See Methods for reproducibility
of experiments.
AP-1 proteins (JUN, FOSL1, JUND) in the nuclei of MDA-MB-231
cells (Fig. 4e). Similar results were obtained in A549 and HCT116
cells (Supplementary Fig. 5b). These interactions were confirmed at
the biochemical level: endogenous FOSL1, JUN and JUND robustly
co-immunoprecipitated with Flag-tagged TEAD1 (Fig. 4f). Finally,
endogenous TEAD1 was co-purified with endogenous FOSL1 and
JUND (Fig. 4g,h and Supplementary Fig. 5c).
It has been recently reported that FOS tethers YAP to the pro-
moter of vimentin, leading to a model in which FOS may directly
recruit YAP/TAZ on DNA independently of TEAD (ref. 23). By co-
immunoprecipitation, we have been unable to detect any protein–
protein interaction between YAP/TAZ and the main AP-1 factors
expressed in MDA-MB-231, that is, JUN, JUND and FOSL1 (Supple-
mentary Fig. 5d–f). By ChIP-seq, the signal of YAP/TAZ peaks, which
matched the TEAD sig nal, did not correlate with that one of JUN
(Supplementary Fig. 5g). Finally, we monitored the capacity of YAP to
activate a luciferase reporter containing polymerized TEAD-binding
sites (8xGT–LUX) or AP-1-binding sites. Despite being artificial, t hese
reporters are highly specific and sensitive, and allow evaluation of
the contribution of individual transcription factors in the absence of
other, and potentially confounding, binding sites (a risk of natural
promoters). YAP could activate 8xGT–LUX but not the AP-1 sensor
(which instead was activated by treatment with the phorbol ester TPA,
an established inducer of AP-1; Supplementary Fig. 5h,i). Collectively,
our results argue against the possibility t hat AP-1 dimers mediate
YAP/TAZ binding to DNA; that said, it remains possible that, in cer-
tain contexts, AP-1 fac tors may also interact with YAP/TAZ to further
enhance the stability of the YAP/TAZ-TEAD and AP-1 complex.
To assess the role of AP-1 in YAP/TAZ/TEAD-mediated
transcription, we generated luciferase reporters containing CTGF and
ANKRD1 regulatory sequences; both contain motifs for TEAD and
AP-1. Mutation of either the TEAD or AP-1 motif reduces luciferase
activity (Fig. 4i), indicating that both sites are required to mediate
YAP/TAZ-dependent transcription. Taken together, the data indicate
that, at YAP/TAZ-bound cis-regulatory elements, YAP/TAZ/TEAD
and AP-1 proteins form a transcription factor complex bound to
composite regulatory elements harbouring both TEAD and AP-1
motifs (Fig . 4j), and jointly regulate gene transcription.
1222 NATURE CELL BIOLOGY VOLUME 17 | NUMBER 9 | SEPTEMBER 2015
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