VGLL4-driven TEAD4 multimerization orchestrates DNA binding and YAP recruitment

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
The Hippo pathway, a highly conserved kinase signaling cascade, is central to regulating cell
growth and proliferation, tissue homeostasis, and organ development. As downstream
effectors of this pathway, TEAD1-4 proteins serve as sequence-specific transcription factors
in mammals. They collaborate with cofactors, such as VGLL and YAP/TAZ, to modulate gene
expression, thereby controlling diverse cellular processes. Here, employing fluorescence-
combined optical tweezers, we demonstrate that monomeric TEAD4 binds to consensus motifs
with association rates significantly higher than nonspecific DNA, while the dissociation rates
are fast and comparable. Yet, TEAD4, through multimerization, gains multiple DNA binding
sites, supporting elongated DNA residence time and YAP recruitment. Moreover, both YAP and
VGLL4 can promote TEAD4 multimerization and strengthen its DNA binding and sequence
specificity. Unexpectedly, the presence of two Tondu domains in VGLL4 elicits a stoichiometry-
dependent effect on YAP recruitment to DNA-bound TEAD4: A low VGLL4:TEAD4 molar ratio
enhances this process, whereas a high ratio inhibits it. These findings offer a dynamic
understanding of how a eukaryotic TF interacts with DNA and underscore a distinct molecular
mechanism by which VGLL4 modulates TNEAD4-mediated YAP recruitment in the Hippo
pathway.

Introduction
The evolutionarily conserved Hippo pathway is one of the cell and tissue growth control mechanisms
that regulate cell proliferation, tissue regeneration, and metastasis1, 2. These processes are mainly
realized by transcription factors (TFs) and coactivators regulating the expression of a variety of target
genes involved3-5. The transcriptional enhanced associate domain (TEAD) TF family and their
transcriptional coactivators Yes-associated protein (YAP) have been identified as key mediators lying
at the heart of the mammalian Hippo pathway6-10. Hippo pathway activation promotes the
phosphorylation of YAP/TAZ by upstream kinases LATS1 and LATS2, which leads to YAP cytoplasmic
retention and transcriptional repression11, 12. Upon dephosphorylation, YAP enters the nucleus and
binds to TEADs, activating the transcription of multiple target genes that regulate cell proliferation,
metastasis, and apoptosis13, 14. Nevertheless, VGLL4 competes with YAP for TEAD binding and
functions as a transcriptional repressor to inhibit organ overgrowth15-18. Moreover, mounting evidence
has suggested the pivotal roles of TEADs in tumorigenesis19, 20. Thus, beyond their function as TFs,
TEADs act as critical oncogenic effectors and represent potential therapeutic targets in cancers.
As a group of key TFs in the Hippo pathway, the TEAD proStein family consists of four members
(TEAD1-4) in mammals, which are widely expressed in diffeErent tissues with both common and distinct
functions21. TEAD1-4 share conserved functional doPmains: an N-terminal TEA/ATTS DNA-binding
domain recognizing the promoters of target genes and a C-terminal protein-binding domain
associating with proteins like YAP and VGLL22-25. TEAD4 is a key member of the TEAD family and
modulates the expression of distinct gene sets to affect different biological and disease processes26.
Notably, TEAD4 functions in Rboth embryonic development and tumorigenesis, which differs from the
other three TEADs27, 28. Its upregulation has been closely linked to multiple malignancies, such as
colorectal cancer29, gastric cancer30, breast cancer31, and prostate cancer32. Extensive studies have
been devoted to understanding TEAD4–DNA interactions at the molecular level. TEAD4 was reported
to specifically bind to the sequence: 5’-TGGAATGT-3’, which is located in the promoter–enhancer
regions of cardiac, smooth, and skeletal muscle-specific genes, named muscle-CAT (MCAT)
elements33, 34. Structural studies have elucidated how the α3 helix in the N terminus of TEAD4
mediates sequence-specific DNA contacts35. Besides DNA sequences, TEAD concentration may add
an additional layer of complexity in controlling its DNA binding activities36, 37. For example, recent
studies have suggested that a high amount of eukaryotic TFs may form biomolecular condensates on
DNA that account for the spatial and temporal organization of transcription38, 39. Additionally,
considering that transcriptional cofactors, such as VGLL1-4, exert their transcriptional functions
through pairing with TEADs, they possibly play a regulatory role in TEAD4’s DNA-binding dynamics
as well40. Particularly, unlike VGLL1-3 that share one conserved TEAD-interacting Tondu (TDU)
domain, VGLL4 contains two partially conserved TDU domains at its C terminus and thus can form a
complex with TEAD4 at a 1:2 molecular ratio15. Therefore, TEAD4 presents a compelling TF model
system given its dependence on DNA sequence, concentration, and cofactors, and established roles
in development and cancer41. A comprehensive investigation of TEAD4’s interplay with DNA and
cofactors at the molecular level would furth er our understanding of the Hippo pathway and inform
potential therapeutic strategies for cancers.
In this work, we combinRed optical tweezers with confocal microscopy and examined the binding
kinetics and profiles of TEAD4 on a single DNA molecule. These characteristics depend not only on
DNA sequence but also on protein concentrations. Notably, TEAD4, through multimerization, extends
DNA residence time, modulates sequence specificity, and facilitates YAP recruitment. Moreover, the
presence of YAP or VGLL4 promotes TEAD4 multimerization and regulates its DNA binding activities,
thereby strengthening a sequence-dependent DNA binding profile under a wide range of protein
concentrations. Surprisingly, VGLL4 binding to TEAD4 exerts a dose-dependent, two-stage effect on
YAP recruitment. Our findings offer detailed DNA binding kinetics of TEAD4 and unravel the regulatory
role of VGLL4 in modulating TEAD4–DNA interactions and YAP recruitment. This mechanistic insight
advances our understanding of how TEAD4 collaborates with cofactors to contribute to transcriptional
outputs in the Hippo signaling pathway.

Results
Characterizing TEAD4 DNA binding kinetics at the single-molecule level
We first sought to explore the dynamic association and dissociation of TEAD4 with DNA at the single-
molecule level. To this aim, we used dual-trap optical tweezersS combined with confocal microscopy to
suspend a DNA molecule between two polystyrene beRads, while monitoring the dynamic interplay
between enhanced green fluorescent protein (eGFP )-fused TEAD4 proteins (TEAD4-eGFP) and the
DNA (Fig. 1a-c). This single-molecule experiment was performed in a multi-channel microfluidic
system, which allows for the quick formaLtion of a single DNA tether (in beads and DNA channels) and
the swift switching of experimental conditions (in protein-containing and protein-free channels) (Fig.
1b)42. The confocal laser repeatedly scanned along the suspended DNA, yielding the binding and
unbinding signals of TEAD4-eGFP in real time (Fig. 1c).
To examine the differences in the binding kinetics of TEAD4 on MCAT and nsDNA, we
constructed a 19-kbp DNA template containing a single MCAT motif located right in the middle of the
DNA template (hereafter referred to as 1× MCAT template) (Fig. 1c and Supplementary Fig. 1). We
started the experiment at an extremely low TEAD4 concentration of 0.1 nM to encourage single protein
binding. As demonstrated by the representative kymograph (Fig. 1d), the monomeric TEAD4-eGFP
binding and unbinding events were immediately noticed along the 1× MCAT DNA in the protein channel.
As expected, TEAD4 displayed a sequence-dependent DNA binding characteristic, with a preference
for the MCAT motif. This sequence-dependent DNA binding may arise from distinct structural
configurations adopted by TEAD4 on nsDNA versus MCAT, as predicted by AlphaFold3
(Supplementary Fig. 2a, b). However, similar to nsDNA, a single TEAD4 protein did not reside on
MCAT for a longer time and dissociated from the motif within a few seconds (Fig. 1d). Our gel-based
bulk assays excluded the possibility that the binding activities were due to the fluorescence labeling
(Supplementary Fig. 3). Given the low protein concentrations used, the short residence times are
also unlikely because of facilitated dissociation43. To quantitatively determine the key kinetic
parameters of TEAD4 binding to DNA, we revisited the experiments under different TEAD4
concentrations and derived their real-time b inding trajectories from the kymographs (Fig. 1d-h and
Supplementary Fig. 4). As reflected by the distinct binding frequencies per 1 kb, the association rate
constant (k ) on MCAT is 200-foTld higher than that on nsDNA (Fig. 1e, f, and h). In contrast, according
on
to the statistical analysis, the residence times of TEAD4 on MCAT and nsDNA are 2.6 s and 3.1 s,
respectively. These data indicate negligible differences in the dissociation rates (k ) (Fig. 1e, g, and
off
h). Moreover, upon binding, a few TEAD4 proteins were found to diffuse on the DNA before
dissociation (Supplementary Fig. 5).
Taken together, we conclude that monomeric TEAD4 realizes the sequence-dependent DNA
binding mainly through varied association rates on different targets, whereas the dissociation process
plays a minimal role in that.
TEAD4 alters DNA binding kinetics through multimerization
The short residence time of TEAD4 on both MCAT and nsDNA raised the possibility that it may employ
a different DNA-interacting mechanism to prolong DNA binding for transcriptional activation. Given
that TEAD4 can form foci in vivo44, 45 and that AlphaFold3 prediction suggests TEAD4–TEAD4
interactions via the TEA domain (Supplementary Fig. 2c), we reasoned that increasing TEAD4
concentration might alter its multimeric states and likely DNA binding kinetics. To test this hypothesis,
we chose to use the 48.5-kbp λ DNA, which contains two MCAT consensus motifs as well as several
MCAT-like motifs. The long stretch of λ DNA offers sufficient binding sites and clear fluorescence
signals for the analysis of both DNA binding kinetics and profiles. To promote protein multimerization,
we incubated the suspended λ DNA in the channNel containing relatively high concentrations of TEAD4
proteins (1, 5, and 10 nM) for 1 min, followed by transporting the tether to a protein-free channel (Fig.
2a). This transportation permits quick dissociation of unstably bound TEAD4 and better fluorescence
visualization owing to the lack oTf background signals (Supplementary Fig. 6a). To our surprise, we
recorded distinct long-liveAd DNA-binding events that can last for tens of seconds in this channel (Fig.
2b). Moreover, upon elevating TEAD4 concentration in the protein-containing channel, a progressive
increase in both long-lived binding events and the residence time is noticeable in the protein-free
channel (Fig. 2b).
To comprehensively understand how TEAD4 exhibited distinct DNA-binding behaviors under
high concentrations, we next performed quantitative analysis on these long-lived DNA-binding events.
We first analyzed their DNA residence times under different TEAD4 concentrations (Fig. 2c and
Supplementary Fig. 6c, d). To differentiate from unstable DNA binding, we only calculated the events
that still stand after 10 seconds. These time distributions were found to follow an exponential decay,
and fitting results showed the residence time increased with the protein concentration (Fig. 2c).
Please note that this calculated residence time is underestimated as the tether transportation time was
ignored. Moreover, quantification of the fluorescence intensity and comparison with a single eGFP
revealed that individual binding events contain variable numbers of TEAD4 molecules, indicating that
a cooperative binding mechanism (Fig. 2d and Supplementary Fig. 7). Remarkably, a positive
correlation between the fluorescence intensity and DNA residence time corroborated that TEAD4
indeed strengthens DNA binding through multimerization (Fig. 2e). To further verify the cooperative
binding, we analyzed the overall fluorescence inNtensity of stably bound TEAD4-eGFP under varying
protein concentrations, which revealed a sig moidal binding curve with a Hill coefficient of 2.7 ± 0.4
(Fig. 2f and Supplementary Fig. 6a, b). Notably, through examining the initial DNA binding states
and conducting mass photometrTy analysis, it is obvious that TEAD4 can self-assemble into a complex
before binding to the DNA (Supplementary Fig. 8c).
Collectively, TEAD4 shifts from monomeric binding to cooperative multimerization at high
concentrations, through which it achieves sustained interactions with target DNA regions. Hereafter,
TEAD4 multimerization is determined based on long-lived DNA binding (over 10 s) together with a
fluorescence intensity exceeding that of a single eGFP.
Sequence- and concentration-dependent DNA binding profiles of TEAD4
Having demonstrated that TEAD4 can prolong the residence time on DNA through multimerization,
we next investigated the regulatory role of DNA sequence in these long-lived DNA-binding events. To
this end, we revisited the single-molecule assay and suspended the 48.5-kbp λ DNA through a
streptavidin-coated bead with a diameter of 4.34 μm and an anti-digoxigenin-coated bead with a
diameter of 2.08 μm (Fig. 2g). The distinct sizes of the beads provide a means to distinguish the
orientation of the DNA and thus allow for the alignment of the TEAD4 binding positions with DNA
sequences (Supplementary Fig. 9a)47. Meanwhile, we employed the position weight matrix (PWM)
of MCAT motifs to predict the binding probability of TEAD4 on each position along the λ DNA and
generated the binding energy landscapes for different DNA sequences (Fig. 2h and Methods)47.
Consistently, we monitored the long-lived multimerized TEAD4 signals on the λ DNA in the protein-
free channel, which increased with the prNeincubated protein concentration (Fig. 2g and
Supplementary Fig. 9b). Analyzing the fluor escence intensity allowed us to acquire the DNA binding
profiles of multimerized TEAD4 (Fig. 2i and Supplementary Fig. 9b). By correlating the measured
DNA binding profiles with the calculated binding energy landscape, we obtained the Pearson
correlation coefficients (PACC) for each experimental condition, which were used as a key parameter
to define the extent of TEAD4 sequence specificity hereafter (Fig. 2i and Supplementary Fig. 9b)47.
This coefficient exhibited a bell-shaped dependence on TEAD4 concentration, with a maximal one
reached at 5 nM (Fig. 2j). By subtracting the fluorescence profile at 10 nM TEAD4 from that at 5 nM
TEAD4, we excluded the possibility that the drop in sequence specificity at 10 nM is solely due to
increased foci size (Supplementary Fig. 9c). These findings suggest that the sequence specificity of
TEAD4 can be significantly altered by varying the protein concentration. At moderate TEAD4
concentrations, DNA sequences dominate its association preference, whereas at higher
concentrations, TEAD4 stabilizes interactions with suboptimal DNA sites through multimerization,
thereby diminishing sequence specificity.
Overall, our findings demonstrated the sequence- and concentration-dependent DNA binding
profiles of TEAD4, with optimal sequence specificity observed at moderate protein concentrations.
MCAT clusters promote TEAD4 multimerization under low concentrations
Given that elevating concentration promotes TEAD4 multimerization for prolonged DNA binding on
nsDNA, it is conceivable that a series of adjacent MCAT motifs, as found in the human genome (chr9:
76,175,201–76,175,280 and 70,038,155–70,038,667), may serve the same purpose at low protein
concentrations. To test that, we constructed a 19-kb p DNA template containing 10 consecutive MCAT
motifs (10x MCAT template) (Fig. 3a and Supplementary Fig. 10). When incubating this DNA
template in a 0.25 nM TEAD4 channel, we monitored long-lived TEAD4 binding events at the MCAT
motifs as expected (Fig. 3a). These observations became more pronounced in a 0.5 nM TEAD4
channel (Fig. 3a). Quantitative analysis revealed a nearly three-fold extension of the residence times
on the 10× MCAT sites than those on the 1× MCAT sites (Fig. 3b, c and Supplementary Fig. 11a).
To address whether these observations are due to TEAD4 multimerization, we collected the photon
numbers per binding event at 0.5 nM TEAD4. It turned out that this number at 10× MCAT sites was
significantly higher than that at 1× MCAT sites (Fig. 3d). Upon elevating the TEAD4 concentration to
5 nM, the markedly enhanced fluorescence intensity was monitored at 10× MCAT sites
(Supplementary Fig. 11b, c). These analyses are in favor of multiple TEAD4 proteins at MCAT
clusters. Moreover, we also noticed that some of the proteins diffused around the clusters as a complex,
supporting that these proteins indeed multimerized at complexes (Supplementary Fig. 11d).
Collectively, these results support that MCAT clusters present a versatile platform for the
assembly of TEAD4 multimer at low protein concentrations. The extended residence times further
support cooperative binding behavior at tandem MCAT motifs.
DNA unzipping unveils multiple DNA-binding sites of multimeric TEAD4
We next aimed to elucidate the underlying mechanism by which TEAD4 multimerization strengthens
DNA binding. The prolonged DNA binding of multimeric TEAD4 could be explained by two conceivable
mechanisms. In one, TEAD4 may enhance its DNA inteEraction strength through multimerization.
Alternatively, multimeric TEAD4 may possess multiple DNA binding sites with comparable interacting
strengths, preventing it from quick dissociation. To differentiate that, we employed an optical tweezers-
based DNA unzipping approach to mechanically separate double-stranded (ds) DNA bound by either
monomeric or multimeric TEAD4 at the MCAT motif (Fig. 4a)48. This approach allows for the accurate
and precise determination of the positions and the strength of DNA–protein interactions (Fig. 4a)49.
The strengthened interaction would result in an increased disruptive force, whereas multiple
interactions would lead to the generation of a set of disruptive forces (Fig. 4b). We constructed a T-
shaped DNA template consisting of two arms and a trunk, with 1× or 10× MCAT motifs embedded in
the middle of the trunk (Supplementary Fig. 12)50. This template was suspended between an optically
trapped bead and a glass coverslip surface via two specially labeled arm ends (Fig. 4a). Monomeric
or multimeric TEAD4 was supposed to bind to the MCAT motif, which is flanked by relatively long
segments of DNA (Fig. 4a). Moving the coverslip away from the optical trap resulted in the unzipping
of the double helix of the DNA trunk, which had a sequence-dependent baseline of approximately 15
pN. The difference between the measured force and that of naked DNA serves as a signal of TEAD4–
DNA interactions51.
We adjusted the experimental conditions to ensure a relatively high probability of monomeric
TEAD4 binding to the single MCAT site. The unzipping force was initially similar to that of the
corresponding naked DNA. However, as the unzipping fork encountered the MCAT motif, the detected
forces rose above those of the naked DNA baseline for all examSined traces, after which the forces
continued to be similar to those of the corresponding nakEed DNA (Fig. 4c). Among the traces, the
single rise in force occurred exclusively around the 1× MACT motif, which was thus attributed to the
disruption of TEAD4–MCAT interaction. We carefully analyzed the strength of the interactions on a
single MCAT site, as indicated by the force peaks. As expected for a thermally activated off-equilibrium
process, the magnitude of the single peak rise in force varied from trace to trace and averaged 23.4 ±
1.6 pN (mean ± SD) (Fig. 4d). We then performed unzipping experiments using the 10× MCAT
template to promote TEAD4 multimerization at MCAT sites. Unlike the single disruptive force peak
with the 1× MCAT template, the force traces often displayed a few rounds of rises and falls in the
disruptive force on the 10× MCAT sites (Fig. 4e, f). The number of disruptive force peaks varied
randomly, with an average number of four, indicating that TEAD4 binds to the 10× MCAT sites in a
non-uniform manner. To address whether TEAD4 binds to the 10× MCAT sites as multimers or as
densely packed monomers, we quantified the DNA base pairs disrupted between adjacent force peaks
(Supplementary Fig. 13a). The averaged 19-bp DNA spacer on 10x MCAT is smaller than the 25-bp
footprint of a single TEAD4 on a MACT motif, ruling out the possibility that TEAD4 monomers bind to
the consecutive MCAT motifs (Supplementary Fig. 13b-e). Considering that the number of TEAD4
molecules within each multimer also varies, we interpret these observations as stochastic multimeric
assemblies of TEAD4 rather than a well-defined, coordinated oligomeric state. Quantitative analysis
revealed an average peak force of 20.2 ± 3.3 pN (mean ± SD), which is slightly less than the single
peak force detected at 1× MCAT (Fig. 4d, g).
Thus, TEAD4, through multimerization, gains multiple DNAS binding sites, with each maintaining
comparable strength, and avoids the quick dissociation of tEhe multimeric TEAD4 from DNA (Fig. 4h).
Cofactors modulate DNA binding kinetics and m ultimeric states of TEAD4
After fully comprehending the DNA binding kinetics and profiles of TEAD4, we next addressed whether
its essential cofactors, such as YAP and VGLL4, could play a regulatory role in that. We first examined
the DNA-binding kinetics of individual TEAD4 proteins in the presence of YAP. On the 1× MCAT DNA
template, the involvement of YAP in the single-molecule imaging assays selectively altered the DNA
binding kinetics of monomeric TEAD4-eGFP proteins. While attenuating TEAD4’s DNA association
rates, YAP specifically prolonged monomeric TEAD4’s residence time on the MCAT motif
(Supplementary Fig. 14a, b). Truncated two TDU domains of VGLL4 (hereafter referred to as 2TDU)
also affect the monomeric TEAD4 binding to DNA in a way similar to that of YAP, and yet they barely
alter TEAD4’s residence time on the MCAT motif (Supplementary Fig. 14a, b).
We next asked whether these two cofactors could also affect the DNA-binding behavior of
multimeric TEAD4. After the pre-incubation of 5 nM TEAD4-eGFP with 20 nM YAP, we loaded them
onto a single λ DNA in the protein channel for 1 min, followed by the fluorescence visualization in the
protein-free channel (Fig. 5a-c). In this channel, we monitored multimeric TEAD4 on DNA with
markedly extended residence times (Fig. 5c, d). Analysis of the fluorescence intensity of each binding
multimer revealed an apparent increase in the number of multimerized proteins upon the addition of
YAP (Fig. 5e), and this promotion was found to be dependent on YAP concentration (Supplementary
Fig. 14c). Additionally, more than one YAP molecule existed in each DNA-bound TEAD4 multimer
(Supplementary Fig. 15a, c). Experiments with a TEAD-binding-deficient YAP mutant, YAP-S94A,
allowed us to attribute these observations to the direct YAP–TEAD4 interaction (Fig. 5a and d, e)52.
As demonstrated by the structural prediction, it iNs likely that YAP reshapes TEAD4’s configuration on
DNA, which encourages TEAD4–TEAD4 inte ractions (Supplementary Fig. 2d). We further examined
the sequence dependence of these DNA-binding events. Based on the obtained PCC, the presence
of YAP augmented the sequeTnce-specificity of TEAD4 under most concentrations (Fig. 5f and
Supplementary Fig. 14dA). Moreover, by quantifying the overall fluorescence intensity of TEAD4-
eGFP on λ DNA, we found that YAP also enhanced DNA binding, which is possibly realized through
the promotion of TEAD4 multimerization (Fig. 5g).
We next addressed whether and how VGLL4 influences the multimeric TEAD4 on DNA binding.
Notably, similar to YAP, the truncated VGLL4 (2TDU) also enhanced TEAD4 multimerization and
extended its DNA-binding duration (Fig. 5a-e and Supplementary Fig. 14c). Previous studies
suggested that the two TDU domains are indispensable in gathering TEAD444. To verify that, we
prepared one TDU domain (1TDU) of VGLL4 (Fig. 5a). Indeed, 1TDU was found to exert minimal
effects on TEAD4, underscoring the critical role of VGLL4’s tandem TDU domains in regulating DNA-
binding kinetics and multimerization of TEAD4 (Fig. 5d, e). It is also noteworthy that 2TDU also
enhances sequence specificity and overall DNA association of TEAD4 (Fig. 5f, g, and
Supplementary Fig. 14e). Similar to YAP, there are a few 2TDU proteins within each TEAD4 multimer
(Supplementary Fig. 15b, d).
Collectively, both YAP and VGLL4 modulate the DNA bindSing kinetics of monomeric TEAD4,
promote TEAD4 multimerization for prolonged residence timEes on DNA, and strengthen the sequence-
specificity of TEAD4 DNA binding (Fig. 5h).
VGLL4 regulates YAP recruitment in a stoichiometry-dependent manner
YAP and VGLL4 are acknowledged Lto compete for TEAD4 binding, which leads to different
transcriptional outputs15, 16. Since we demonstrated that both YAP and VGLL4 can modulate TEAD4
DNA binding, we next asked how they compete with each other for DNA-bound TEAD4. To this aim,
we first prepared the mCherry-fused YAP protein (YAP-mCherry) and explored how it was recruited
by TEAD4 proteins alone. Our single-molecule experiments revealed a low YAP-mCherry recruitment
efficiency by DNA-bound TEAD4 (Supplementary Fig. 16a). To provide a desired platform for stable
YAP binding, we chose to saturate TEAD4 on a single λ DNA molecule by suspending it in a 50 nM
TEAD4 channel, after which the tether was transferred to a 25 nM YAP-mCherry channel for
fluorescence visualization (Fig. 6a). In this experimental condition, we detected stable binding of YAP-
mCherry to the TEAD4-coated DNA (Fig. 6b). Control experiments verified that this binding activity is
due to YAP–TEAD4 interactions instead of YAP–DNA interactions (Supplementary Fig. 17a). We first
examined whether VGLL4 could outcompete YAP for TEAD4 binding. Our single-molecule assays
demonstrated that the presence of 2TDU indeed accelerated the dissociation of YAP from DNA-bound
TEAD4, in well agreement with previous studies (Supplementary Fig. 18)15, 16.
Next, we addressed how VGLL4 affects YAP recruitment to the DNA-bound TEAD4. We
suspended the DNA tether in a mixture of TEAD4 and 2TDU before loading YAP-mCherry.
Unexpectedly, a titration of 10 nM 2TDU into 50 nM TEAD4 allowSed for the observation of enhanced
YAP-mCherry binding (Fig. 6b). Nevertheless, further elevEating the 2TDU concentration to 100 and
500 nM gave rise to an opposite effect on YAP recruiPtment (Fig. 6c). Other than simple competition,
our findings support a ratio-dependent, two-stage effect of 2TDU on YAP recruitment to DNA-bound
TEAD4. It is reasonable to speculate that at low concentrations, 2TDU promotes TEAD4
multimerization and DNA binding, subsequently facilitating YAP recruitment. However, 2TDU, at high
concentrations, completely oRccupies the YAP binding domain of TEAD4 (TEAD4-YBD), blocking YAP
binding to the DNA-bound TEAD4-2TDU complex.
To provide additional evidence for this hypothesis and better understand the promotion and
inhibition of YAP recruitment under varied 2TDU concentrations, we labeled 2TDU with LD655 (2TDU-
LD655) and confirmed its inability to directly bind to DNA (Supplementary Fig. 17b). By
simultaneously recording the fluorescence signals of both 2TDU-LD655 and YAP-mCherry, we found
the low 2TDU-LD655 recruitment efficiency by DNA-bound TEAD4 and thus elevated the TEAD4
concentration to 50 nM to amplify the fluorescence signal of 2TDU-LD655 (Supplementary Fig. 16b).
Under a low 2TDU:TEAD4 molar ratio, we observed a limited number of 2TDU-LD655 foci along the
DNA (Fig. 6d). As expected, some of the detected YAP-mCherry foci colocalized with those of 2TDU-
LD655 on DNA, supporting that 2TDU promotes TEAD4 multimerization for YAP recruitment (Fig. 6d).
However, in sharp contrast, under a high 2TDU:TEAD4 ratio, YAP-mCherry signals were dramatically
diminished along the DNA, even though the number of 2TDU-LD655 foci significantly increased under
this condition (Fig. 6e). These findings indicate that a sufficient amount of 2TDU proteins indeed
outcompetes YAP for TEAD4 binding. We also determined the protein ratio within the complexes,
which further corroborated that the 2TDU binding to TEAD4 exerts a dose-dependent, two-stage effect
on YAP recruitment. (Supplementary Fig. 19). After transferring the DNA tether into a channel
containing TEAD4 and YAP-mCherry, we detecteNd YAP-mCherry binding signals at the very beginning
of scanning, indicating that YAP likely pre-ass embles with TEAD4 multimers in solution before binding
to DNA (Supplementary Fig. 20).
In summary, our findingRs unveil a ratio-dependent modulation of YAP recruitment to DNA-bound
TEAD4 by VGLL4 and underscore a subtle balance between TEAD4 multimerization and coactivator
competition induced by VGLL4.

Methods
Protein expression and purification
TEAD4 and TEAD4-eGFP. The human TEAD4 gene was cloned into pET28a with a His -tag at the N
6
terminus and expressed in Escherichia coli (E. coli) BL21(DE3) (TransGen). Protein expression was
induced by 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) until OD =0.8 in Kanamycin
600
resistance-containing LB medium was cultured for an additional 16 h at 18 °C. Cells were then
harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl,
5% glycerol, and 1% protease inhibitor). The cells were lysed by a pressure homogenizer at ∼750 bar
five times. The lysed solution was then centrifuged at 13,500 x g for 30 min. The soluble fraction with
lysis buffer was loaded onto Ni-NTA Agarose (TransGen) and then was further washed with lysis buffer
containing different imidazole concentrations of 20, 50, 100, and 300 mM. The proteins were eluted
with 300 mM imidazole elution buffer. The eluate was then concentrated using 30-kDa filtration to a
certain volume at 4°C. The protein was further purified with anion exchange chromatography using a
5-ml HiTrap Q HP column. Ultimately, the proteins were buffer-exchanged into a storage buffer (20
mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol) and stored at −80°C before use. TEAD4-eGFP was
expressed and purified similarly.
YAP, YAP-S94A, and YAP-mCherry. The human YAP gene was cloned into pET28a with a His -tag
6
at the N terminus. The methods of protein expression and purification were the same as above. The
protein was eluted with 300 mRM imidazole elution buffer, and the protein was further purified with gel
filtration chromatography with a Superdex 200 increase 10/30 column (GE Healthcare) in storage
buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol). YAP-mCherry was expressed and purified
similarly.
2TDU, 2TDU-LD655, and 1TDU. The human 2TDU gene was cloned into pET28a with His -tag and
6
a SUMO tag at the N terminus. The methods of protein expression and purification are as
aforementioned. The protein was purified with gel filtration chromatography with a Superdex 200
increase 10/30 column (GE Healthcare) in storage buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 5%
glycerol). Peak fractions containing sumo-2TDU were pooled and then added SUMO protease with
reaction buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 5% glycerol) at 4 °C overnight. The mixed
solution was passed over Ni-NTA resin to remove any contaminating SUMO tag and His -sumo
6
protease. The 2TDU protein was finally concentrated and changed to the storage buffer (20 mM Tris-
HCl pH 8.0, 500 mM NaCl, 5% glycerol) using a 3-kDa Centrifugal Filter to a certain volume at 4°C.
We used LD655 NHS ester (Lumidyne Technologies) to nonspecifically label the primary amines
of TEAD4. Preferential N-terminal labeling was achieved by labelSing at low pH (7.0) for an NHS ester
reaction63. Labeling was performed with 50 mM HEPES pHE 7.0, 150 mM NaCl, 1 mM DTT, and 0.25
mM EDTA. 2TDU was incubated with LD655 dye at a P1:5 molar ratio for 1 h at room temperature, and
the reaction was quenched with 25 mM Tris-HCl pH 6.8 for 5 min. Excess dye was removed from
2TDU by buffer exchange with storage buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol).
The final labeling ratio was estimated to be ∼60%. The final 2TDU-LD655 was aliquoted and stored
at -80°C.
The 1TDU peptide (sequence: DPVVEEHFRRSLGKNYKE) was commercially synthesized
(Sangon Biotech) based on the TDU domain of VGLL4. The peptide was purified by high-performance
liquid chromatography (HPLC) and verified by mass spectrometry to ensure its purity and sequence
accuracy. Prior to use, the peptide was dissolved in ddH O and stored or diluted to the desired
2
concentration for experiments.
Preparation of DNA templates
1× and 10x MCAT DNA Templates. The MCAT DNA template for the optical tweezers-based single-
molecule assays consisted of two DNA segments (L2 and L1/L3). The 9.5-kbp 1× MCAT DNA L1 was
produced by PCR-amplifying the recombinant pET28a plasmid using 5’ end labeling biotin primer R
and containing the MCAT sequence primer F. The 9.6-kbp L2 DNA segments were polymerase chain
reaction (PCR)-amplified from the pET28a-MCAT plasmid using 5’ end labeling biotin primers. The
final 1× MCAT DNA was produced by ligating the L1 and L2 DNA segments at a 1:1 ratio using T4
DNA ligase (NEB) (Supplementary Fig. 1). The 10× MCAT DNA segment was obtained by annealing
two oligonucleotides in the annealing buffer (20 mM Tris-HCl pH 8.0 and 100 mM NaCl)
(Supplementary Table 1). The 10× MCAT DNAN segment was inserted into the recombinant pET28a
(9.6 kbp) to create a new plasmid (pET28a-M CAT plasmid) (Supplementary Fig. 10). The 9.5-kbp L2
and 9.6-kbp L3 DNA segments were PCR-amplified from the pET28a-MCAT plasmid using 5’ end
labeling biotin primers. The resTulting DNA fragments were digested with XbaI (NEB) to create an
overhang for ligation. The final product was produced by ligating the L2 and L3 DNA segments at a
1:1 ratio using T4 DNA ligase (NEB).
The λ DNA Template. The construction of the λ phage DNA template followed a previously described
method64. In brief, biotinylated λ DNA was generated by labeling the 3’ ends through the extension of
5’-overhangs using an exo-Klenow fragment. The reaction was carried out by incubating 10 nM λ DNA
(Thermo Scientific) with 250 μM dGTP/dATP/dTTP, 10 μM biotin-14-dCTP (Thermo Scientific), and 5
U Klenow exo– (Thermo Scientific) in a reaction buffer containing10 mM Tris-HCl pH 7.9, 50 mM NaCl,
10 mM MgCl₂, and 1 mM DTT at 37°C for 1 h, followed by heat inactivation at 75°C for 10 min.
The λ DNA template with one end biotinylated and the other end digoxigeninated was constructed
through annealing 3’ end labeling biotin or digoxigenin oligonucleotides (Sangon Biotech) to 5’
overhangs. The oligonucleotides are also labeled 5’ phosphate, ligated to the phosphate backbone of
λ DNA using T4 ligase (NEB) to avoid nicks.
The T-shaped DNA template. The T-shaped DNA template for the single-molecule unzipping assay
consisted of three DNA segments (two arms and a trunk) and waSs prepared as previously described
(Supplementary Fig. 12)50. Briefly, Arm 1 was PCR-aEmplified from plasmid pBR322 using a
digoxigenin-labeled primer. The resulting DNA fragmePnt was digested with BstXI (NEB) to create an
overhang and was subsequently annealed to a short DNA with a complementary overhang formed by
Adapter 1 and Adapter 2. Arm 2 was PCR-amplified from plasmid pBR322 using a biotin-labelled
primer. The resulting DNA fragment was digested with BstXI (NEB) to create an overhang and was
subsequently annealed to adRapter 3 and adapter 4. Adapter 2 from Arm 1 and Adapter 4 from Arm 2
were partially complementary to each other and were annealed to create a short 35-bp trunk with a 3-
bp overhang for trunk ligation. The 0.5-kbp trunk containing the MCAT motif sequence was amplified
from the plasmid pEGFP-N1 and digested with AlwNI (NEB). The final product was produced by
ligating the arms to the trunk at a 1:4 ratio. All DNA products were purified with a SanPrep Column
PCR Product Purification Kit (Sangon Biotech).
Optical tweezers-based single-molecule assays
A dual-optical tweezer setup combined with confocal microscopy and microfluidics (LUMICKS, C-trap)
was used to perform the single-molecule assays65, 66. In brief, a 1064-nm fiber laser and a water-
immersion objective were used to create two orthogonally polarized optical traps. The trap separation
was controlled using a piezo mirror for beam-steering one trap. Force measurements were performed
by back-focal plane interferometry of the condenser top lens using a position-sensitive detector. A
computer-controlled stage enabled rapid movement of the optical traps within a multiple-channel flow
cell64. This microfluidic system allowed for the rapid in situ construction and characterization of DNA
dumbbell constructs, and facilitated the swift transfer of tethered DNA between different flow channels
(Fig. 1b). In the assay, a single DNA molecule was initially captured between two streptavidin-coated
polystyrene beads (4.34 μm in diameter, Spherotech) or between one streptavidin-coated polystyrene
bead and one anti-digoxigenin-coated bead (2.01 μm in diameter, Spherotech) using the multichannel
laminar flow cell and was tensioned by in creasing the distance between the optical traps. The
presence of a single suspended DNA molecule was verified by its inherent mechanical force–
extension relationship67. The DTNA tether was then transported to the protein or buffer channel as
described for each assaAy. In these channels, the 5.8-μm (19-kb DNA) or 15-μm (43.5-kb DNA)
constant distance was applied to the DNA tether via a high-frequency feedback system on the steered
trap during data acquisition. Unless stated otherwise, all experiments were carried out in reaction
buffer containing 20 mM HEPES-KOH pH 8.0, and 100 mM NaCl.
Confocal scans were performed when DNA tethers were in channels 3 and 4. A 488 nm laser
was used for imaging TEAD4-eGFP. Kymographs were generated via a confocal line scan through the
center of the two beads with a pixel size of 75 nm for 1 ms when imaging with TEAD4-eGFP alone. A
561-nm laser and a 638-nm laser were used for YAP-mCherry and 2TDU-LD655 visualization with a
scanning pixel size of 75 nm and a duration of 0.5 ms. In the TEAD4-eGFP, YAP-mCherry, and 2TDU-
LD655 colocalization experiment, the DNA tether was preincubated in a channel containing 10 or 100
nM 2TDU-LD655 and 50 nM TEAD4 under 15 μm for 30 s, then we mimicked the in vivo relaxed status
of the DNA by maintaining the two traps approximately 3 μm apart (~ 0 pN) and waiting for 30 s. After
an indicated relaxation period, we immediately transferred the DNA tether into the YAP-mCherry
channel and moved the steered trap away from the fixed trap at a slow rate of 0.5 μm/s. The confocal
images were acquired at ~10 pN in the YAP-mCherry channel, sequentially illuminating with 638 nm,
532 nm, and 488 nm lasers.
Single-Molecule Fluorescence Data Analysis
Kymographs processing and kinetic analysis. Kymographs were analyzed using the Lakeview
1.2.0-beta.3 provided by LUMICKS. The following parameters were applied for particle tracking:
minimum photon counts of 3, positional search range of 0.20, maximum gap of 6, minimum length of
3, expected spot size of 0.4, and expected velocity of 0. For tracks with gaps that could not be
connected by the default parameters (more commonly observed for long-lived tracks), the 'Connect
line' function was used to manually connect two lines. Notably, all kymographs were acquired without
laminar flow.
We tracked single-molecule binding traces from kymographs to extract residence time and
binding frequency (f ) information (Fig. 1e). The dissociation rate constant (k ) was obtained by
binding off
fitting the resulting histogram to a single exponential decay function using Origin 2023b (Fig. 1e).
The binding frequency characterizes the average number of detected binding events per second
on the 1 kbp range of DNA in the kymograph; n is the number of binding events, and L is the detected
DNA range. The formula for calculating f is provided as:
binding
f (s-1)= n·L (1)
binding
Δt·1000
Under identical experimental conditions, f obtained from multiple traces are averaged. The
binding
average f was then plotted against the corresponding pSrotein concentrations using Origin
binding
2023b, and a linear fit was applied to the data. The slope ofE this linear relationship was interpreted as
the association rate constant (k ).
on
Photon counts calculation for TEAD4 trajectories. To quantify fluorescence signals, the absolute
photon count per pixel for each classifieLd TEAD4 binding trajectory was averaged over 100 frames to
generate a data point using Pylake v.1.6 provided by LUMICKS, an open-source Python-based
framework specifically designed for single-molecule biophysics data processing. Image acquisition
and photon counting analysis were performed following the standardized workflow outlined in the
LUMICKS Pylake documentation. Custom analysis scripts were developed in a Python 3.9
environment, implementing the core methodologies described in the "Photon Counts" section of the
official user guide, where each pixel represents the summed photon counts. Each data point
represents the averaged photon count per frame within a pixel box and correlates with the binding
lifetime of its corresponding TEAD4 trajectory. All graphs and plots were generated using Origin 2023b
for precise data visualization and analysis.
Hill equation fitting. To quantitatively analyze the binding behavior of TEAD4 to DNA, we performed
a Hill curve fitting. The average intensity of TEAD4 on DNA was calculated over a time window of 70
to 90 s to minimize the influence of two potential confounding factors: the immediate drop in TEAD4
signal due to transient binding events and the gradual signal reduction caused by photobleaching
(Supplementary Fig. 6). The average intensity values, corresponding to the reaction equilibrium,
were used as the dependent variable (Y-axis), while the corresponding protein concentrations were
used as the independent variable (X-axis). The Hill equation was then applied to model the cooperative
binding behavior (2), expressed as the equation below, where y represents the average intensity, V
max
is the maximum intensity at saturation, k is the disNssociation constant, and n is the Hill coefficient, which
indicates the degree of cooperativity in the b inding process. This approach allowed us to derive key
binding parameters, including the dissociation constant (k) and the Hill coefficient (n), providing
insights into the affinity and Tcooperativity of TEAD4–DNA interactions under the experimental
conditions. A
y = V
[TEAD4]n
(2)
max kn+xn
Calculation of photon count distributions.
The DNA tether was incubated in the protein channel for 1 min, then transferred to a protein-free
channel for immediate imaging. The first frame of confocal images acquired in the protein-free channel
(pixel size: 75 nm) was used for fluorescence analysis (Pylake v1.6, LUMICKS). The maximum
projection intensity profile along the DNA was determined by summing photon counts within a 12-pixel
region centered on the DNA axis. The lateral range of the selected region was adjusted according to
protein concentration to exclude contributions from the bead boundaries to the photon count
distributions. For each fluorescence image, the calculated photon count distribution was processed in
sequential or reverse order based on bead size to ensure sequence consistency, and the average
photon count distribution for each condition was determined. Each condition included 19-54
independent experiments.
Analysis of TEAD4 sequence-specific binding to λ DNA. TSo assess the specificity of TEAD4
localization on λ-DNA, a previously reported MCAT motif wEas utilized37, 68. The position weight matrix
(PWM) was retrieved from the JASPAR database (PMatrix ID: MA0809.2), consisting of four rows
corresponding to each DNA base and columns representing each position within the motif (Fig. 2h
and Supplementary Table 1). The matrix values reflect the relative frequency of a specific base at a
given position in the motif. Since such matrices are generally derived from genome-wide in vivo
protein-DNA binding data, thRey offer a probabilistic measure of protein binding affinity to specific DNA
sequences.
The computational methodology for analyzing protein–DNA binding specificity in this study was
adapted from established approaches using the PWM, which is denoted as 𝑀̅F for the forward strand
𝑛𝑏
and 𝑀̅R for the reverse strand. It quantifies the relative frequency of base 𝑏 at position 𝑛 when the
𝑛𝑏
protein is bound to DNA. To account for sequences deviating from the consensus, matrix elements
where 𝑀 ≤ 𝑒 was replaced by a minimal value 𝑒, in this case, 𝑒=0.4. The average PWM, 𝑀̅ , was
𝑛𝑏 𝑛𝑏
calculated as:
𝑀̅ =
𝑀̅
𝑛
F
𝑏
+𝑀̅
𝑛
R
𝑏 (3)
𝑛𝑏
2
Where 𝐿 is the sequence length. The binding energy landscape was inferred using:
𝑀̅
𝜖 −𝜖 = 𝑘𝑇ln ( 𝑛𝑏) (4)
𝑏,𝑛 𝑏∗,𝑛 𝑀̅ 𝑛𝑏∗
and the binding energy difference for the sequence 𝐵̅ relative to the consensus was calculated as:
𝛿𝐸(𝐵̅) = ∑ (𝜖 −𝜖 ) (5)
𝑛 𝑏,𝑛 𝑏∗,𝑛
This approach allowed for the inference of binding energy landscapes, as detailed in the related work47.
For each average intensity profile, the correlation with the coarse-grained binding energy profile by
Savitzky–Golay (Points of Window: 4000, Polynomial Order: 3) was quantified as Pearson’s
correlation coefficient. This filter fits successive subsets of adjacent data points with a low-degree
polynomial by least squares regression, therebNy preserving peak shape and height while reducing
high-frequency noise.
Single-molecule DNA unzipping assay and data analysis
DNA unzipping experiments were performed on an M-trap optical tweezer from LUMICKS (Amsterdam,
Netherlands). The sample chamber preparation was described previously51. Briefly, glass coverslips
were cleaned and functionalized with partially biotinylated polyethylene glycol (PEG) (Laysan Bio)69
and then coated with streptavidin (Thermo). Biotin-tagged DNA was added and incubated to form the
DNA tethers. Antidigoxigenin-coated 0.5-μm polystyrene beads (Polysciences) were then added to the
chamber. For the single-molecule DNA unzipping experiment, a T-shaped DNA template flowed into
the chamber immediately before data acquisition. The experiments were conducted in a climate-
controlled room at a temperature of 23.3 °C; however, owing to local laser trap heating, the
temperature increased slightly to 25 ± 1°C70. The experiments were conducted by mechanically
unzipping the dsDNA at a slow velocity of 100 nm/s to probe the potential interactions at the fork.
Single-molecule DNA unzipping data were acquired at 5 kHz and later filtered to 50 Hz. The
acquired data signals were converted into force and DNA extension as previously described51, 71. The
elasticity parameters of both dsDNA and ssDNA for data conversion were obtained from DNA force–
extension curves72. In the unzipping experiments, one separated base pair generated two nucleotides
of ssDNA. Accordingly, the real-time DNA extension in nm was fSurther converted into the number of
base pairs unwound based on the elastic parameters of ssDENA under our experimental conditions. To
improve the positional precision and accuracy, the force-versus-unzipped-base-pair curves were
aligned to the theoretical curve by the cross-correlation of a region before and after the ternary
complex disruption71. To account for minor instrumental drift, trapping-bead size variations, and DNA
linker variations, the alignment allowed for a small additive shift (< 5 bp) and multiplicative linear stretch
(< 2%) using algorithms simiRlar to those previously described71.
Statistical Analysis
To determine the appropriate statistical approach, data were first subjected to normality testing using
the Shapiro–Wilk test. For datasets that conformed to a normal distribution, one-way analysis of
variance (Tukey HSD) was subsequently performed to assess the statistical significance between
groups; otherwise, the Kruskal-Wallis H test was applied. All analyses were conducted using Origin
2023b, and results were considered statistically significant when p < 0.05.
Statistics and Reproducibility
No statistical method was used to predetermine sample size. Experiments were not randomized, and
investigators were not blinded to allocation during experiments or outcome assessment. Statistical
analyses were performed as indicated in the figure legends. All experiments were independently
reproduced at least 3 times, yielding consistent results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, Rend, Zhao et al. used optical tweezers combined with confocal fluorescence microscopy, to study the DNA binding kinetics of the transcription factor TEAD4. The authors found that monomeric TEAD4 binds to its canonical DNA MCAT motif with an association rate significantly higher (~200-fold) than to non-specific DNA, while the dissociation rates were indistinguishable. Most significantly, the authors observed that the TEAD4 alters its DNA binding kinetics and prolong its residence time through multimerization, which is dependent on DNA sequence and concentration of the factor. By using a T-shaped DNA template consisting of two arms and a trunk harboring 1xMCAT or 10xMCAT motifs suspended between an optically trapped microsphere and a glass coverslip, they could generate mechanical force to unzip the DNA double helix trunk. The DNA trunk alone unzipped at ~15 pN, while a single sharp force peak of ~ 23 pN (for the 1xMCAT template), or a series of them of about 20 pN each (for the 10xMCAT template), were associated with the disruption of the TEAD4-DNA interactions for a monomeric or multimeric binding mode, respectively. They also show that both YAP and the truncated VGLL4 (two TDU domains deleted, referred as the 2TDU construct) co-factors modulate the DNA binding kinetics of monomeric TEAD4 by attenuating TEAD4’s DNA association rates and extending the TEAD4’s residence time on the MCAT motif. These factors also promoted TEAD4 multimerization and prolonged its residence times on DNA while enhancing the sequence-specificity of TEAD4 DNA binding. Finally, the authors revealed a molar ratio-dependent mechanism by which VGLL4 (2TDU) affects the YAP recruitment to DNA-bound TEAD4. While at low-concentrations the 2TUD promoted TEAD4 multimerization and DNA binding, facilitating YAP recruitment, at high-concentrations 2TDU outcompetes YAP for TEAD4 binding.\nOverall, the authors have used well-thought-out single molecule biophysical approaches to characterize the TEAD4-DNA binding kinetics and the nature of these interactions under different conditions, propose the idea of induced multimerization, and relate it to the presence of co-factors. While this study improves our knowledge of an important biological system, there are issues that need to be addressed prior to publication.\nMajor points:\n- Have the authors considered the use of AlphaFold to predict how the TEAD domains could engage in the multimerization process? This is a very common practice that has often produced interesting results that can then be tested via mutagenesis. The authors could do this both in the absence and presence of target DNA motifs. They could even attempt to predict the possible structurers of the TEAD-DNA-YAP or TEAD-DNA-VGLL4(2TDU) ternary complexes. Given that the authors have performed experiments with a TEAD-binding-deficient YAP mutant (YAP-S94A) to support that their observations are attributable to direct interactions between YAP and TEAD4, it would greatly strength their study if the authors could add in silico structural predictions.\n- The authors should clarify if the TEAD4 ‘multimerization’ process is precise or if there are different number of molecules of the transcription factor per cluster. Does the size of the cluster depend on the concentration of cofactors? Is there more than one co-factor (YAP or VGLL4) per each TEAD4 cluster?\n- When using DNA fragment harboring 10xMCAT motifs, the authors found that TEAD4 oligomerization was promoted, and four consecutive peaks of force ramp were a common trait during the trunk DNA unzipping. The authors should discuss more about this observation. For instance, what could be a possible explanation for the observed four peaks of force as a recurrent trait even when 10xMCAT motifs are present? Is the TEAD4 multimerization a well-coordinated process, where this final TEAD4 oligomer state is well-defined and possibly also affects the local DNA structure in a defined manner?\n- The authors should rationalize their proposed molecular mechanism in which the monomeric TEAD4 distinguishes specific MCAT motifs from non-specific DNA through accelerated association rates (kon)”. While they state that ‘this rapid and selective association reflects a facilitated target search mechanism, balancing efficient chromatin scanning with sequence-specific recognition’, one would expect that in such a search scenario the cognate binding site would reduce the dissociation rate (koff) of the factor, as it is more typically the case, while they see non-significant differences for this rate between specific and non-specific DNA. Instead, a difference in association rate requires a non-standard explanation in a search mechanism model. How do the authors envision it?\n- Data provided by the authors showed that elevated TEAD4 concentrations lead to TF multimerization, which further stabilizes DNA interactions, even on nonspecific sequences and finally affects the sequence specificity binding. Yet, it is unclear if the multimerization can take place in solution as well, or on the DNA only? The authors could perform some biochemical studies in solution to assess this question. For example, SEC or mass photometry in the range of concentrations tested in the tweezer’s experiments.\n- The authors should clarify if there are ‘clouds’ of MCAT motifs present in the human genome that would lead to the formation of the TEAD4 clusters they see with the 10XMCAT motifs. Also, are the TEAD4 foci formed in vivo, as cited by the authors, possibly related to clouds of MCAT motifs and multimerization of the factor, or are these independent processes in vivo?\n- It is interesting that the activator YAP and the repressor VGLL4 act in a similar manner at the molecular level, both enhancing the TEAD4 binding to DNA by promoting multimerization, in spite of leading to oppose transcriptional outputs. Can the authors speculate in their discussion the possible cellular conditions in which activation or repression will be stablished at a MCAT site? Is the repressive activity of VGLL4 only seen in YAP-responsive genes, so repression is just by competition with YAP only?\nMinor points:\n- During the experiments using the dual-trap Optical-tweezers combined with Fluorescence, did the authors hold the DNA tether at a constant force? They should clarify this condition and also mention if the system behavior was tested at different forces.\n- In line 22 of Page 12, the authors wrote “The effect of truncated two TDU domains of VGLL4 (hereafter referred to as 2TDU) on monomeric TEAD4 binding to DNA was similar to that of YAP (Supplementary Figure 2 S11A-C)”. After inspecting the mentioned figures, particularly S11B, it is not apparent that YAP and 2TDU cause a similar effect on the TEAD residence time. It will be helpful if the authors include the Mean +/- SD values obtained, right on top of the histograms, and explicitly mention if a significant difference was observed or not.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In this manuscript, Ren et al. investigated the DNA binding behavior of the transcription factor TEAD4 and its regulation by cofactors VGLL and YAP. Using single-molecule fluorescence combined with optical tweezers, they found that monomeric TEAD4 binds to consensus motifs with a higher on rate than to nonspecific DNA, while the off rates are comparable. Multimerization of TEAD4 led to longer DNA residence time and YAP recruitment. Both YAP and VGLL4 promote TEAD4 multimerization, thereby boosting its DNA binding and sequence specificity. Finally, VGLL4 elicits a stoichiometry-dependent effect on YAP recruitment to DNA-bound TEAD4. Overall, this is a well-conducted study that provides important insights into the interplay between several key proteins in the Hippo signaling pathway. The data are of high quality and the analyses are rigorous. I would recommend publication after the following points have been addressed.\nMajor points:\n(1) As introduced in the paper, YAP functions as a coactivator of TEAD4, while VGLL4 functions as a transcriptional repressor. But they seem to exert a similar effect on TEAD4-DNA interaction (Figure 5). Nonetheless, Figure 5H gives the impression that YAP and 2TDU modulate TEAD4 binding to DNA through different mechanisms. Please clarify.\n(2) 2TDU was labeled with LD655 as stated multiple times in the text. But in page 25 Line 8: “A 561-nm laser… were used for 2TDU-LD566…” If it’s indeed LD655, it should be excited by the red laser and therefore in the same channel as YAP-mCherry. This inconsistency needs to be addressed.\n(3) In the experiments where 10x MCAT sites were used, the brighter TEAD4 intensities could be due to a single TEAD4 multimer spanning the 10x MCAT site, or multiple TEAD4 units binding close to each other. Figure S9B provides compelling evidence for the former scenario, although the frequency of such behavior is not quantified. On the other hand, the DNA unzipping results in Figure 4 seem to favor the latter scenario (i.e. multiple force peaks correspond to multiple TEAD4 units). Please explain how the latter scenario was ruled out, or perform additional analysis (such as calculating the distance between consecutive force peaks) to provide more definitive evidence.\nMinor points:\n(1) The decrease in sequence specificity of TEAD4 binding at 10 nM (Figure 2J, 5F) is somewhat surprising. Could the calculation of the correlation coefficient be influenced by the size of the TEAD4 foci and/or their diffusivity?\n(2) In Figure 6, lambda DNA was used instead of MCAT-containing DNA. Are there MCAT-like motifs in the lambda DNA sequence?\n(3) Since stoichiometry is suggested to be a key regulatory mechanism for the TEAD4-YAP-VGLL4 interplay, it would be helpful to compare the physiological concentrations of these proteins to those used in this study.\n(4) The first paragraph of Discussion: “This rapid and selective association reflects a facilitated target search mechanism…” The definition of facilitated search typically involves nonspecific binding of a TF followed by 1D diffusion. But TEAD4 diffusion on DNA was only briefly mentioned (Figure S4) and its prevalence was not described.\n(5) In Discussion (page 18), the authors compared the effect of VGLL4 on TEAD-DNA interaction to their homologs in Drosophila. However, Tgi reduces the binding lifetime of Sd as referenced in the paper, while 2TDU increases the residence time of TEAD4. Therefore, the two systems seem to display opposite rather than similar effects.", "reviewer_4": "This manuscript describes how the TEAD4 protein interacts on Lambda DNA and DNA containing the specific TEAD4 motif, repeated 1x and 10x. TEAD4 concentration is varied from very low concentrations (under 1 nM) to examine binding dynamics, specifically on and off rates and mean residency times, as well as at higher concentrations, notably from 1 to 10 nM. Physiologically relevant co-factors, VGLL4 and YAP, are also added to determine their in-vitro system effects, specifically their impact on TEAD4 binding kinetics and sequence localization. The researchers use a confocal optical tweezer system.\nThe main findings are that TEAD4 alone binds DNA, has preference for its specific motif, and can multimerize on DNA, depending on concentration and location. Residency times are increased with increasing concentration, though off rates in the dilute concentration regime (under 1 nM) are comparable, suggesting proteins find their binding targets through on associations (which have differing time constants). The 10x MCAT motif is suggested to facilitate multimeric TEAD4 assemblies with longer residence times. Additionally, addition of co-factors increases TEAD4 residence times, only in their full-length forms and improve sequence specificity.\nThe residence time analysis as well as on and off rates for TEAD4 alone and with co-factors is generally well supported by the data.\nThe main points of clarification are below:\n1) In the main text, TEAD4 sequence specificity is described as being modified and altered (in terms of TEAD4 alone) and augmented (in terms of co-factors). Yet, the graphs and texts do not fully substantiate the claim of modification via concentration, rather support a DNA surface saturation. The text describes the drop in sequence specificity at 10 nM as \"engaging with non-consensus sequences\" but this is not fully defined. What causes the engagement with non-specific DNA?\n2) In Figure S5, kymographs are shown for 1 nM and 30 nM in the protein channel, which are not background subtracted, which makes it difficult to understand in the 1 nM case and impossible to understand in the 30 nM case. It would be helpful to also show the other conditions (5 and 10 nM) in the protein-channel, also as background subtracted. This would be informative for understanding the formation of the multimeric TEAD4 assemblies, whether they assemble on the DNA surface or are formed in the bulk environment, since this discrepancy nor biochemical description of higher order formation are described in the text.\n3) To the definition of multimerization, Figure 2D shows the single eGFP photon count. However, it is unclear if, above this line, the signal is considered from multimerized protein. In Figure S6E, which supports the multimerization definition, the single dCas9-eGFP histogram is being compared with the integrated signal of many TEAD4-eGFP molecules, and does not appear to consider the maximum intensity that could arise from a single layer of eGFP molecules, since the signal will be an integration of intensity from a specific number of base pairs along the DNA surface. Therefore, a clearer definition of multimerization should be included. This is necessary for much of the results discussion, such as during the section that discusses the binding kinetics and profiles of multimeric TEAD4 (Figure 2) and the 10x MCAT producing TEAD4 multimers (Figure 3). For example, are multimeric assemblies really being produced or is the signal 10x higher from 10 TEAD4-eGFP binding subsequently? Is the signal higher than the integration of 10 molecules?\n4) In Figure 3, multimerization is not substantiated. An addition of a line in 3D to show what constitutes bound TEAD4 vs multimer TEAD4 would be helpful.\n5) How fluorescence profiles are determined for the correlation analysis is not clearly found in the text or methods, for example, which time frames are considered and if this profile is produced with images from the protein channel or buffer channel, and whether they consider background subtraction. This would be very helpful in terms of procedural clarity.\n6) TEAD4 PWM is used to calculate the binding energy across the DNA surface to which the fluorescence profile is compared (PCC). It is stated that the energy profile is coarse grained, but not how or to what resolution it is coarse grained. Does the coarse graining significantly alter the energy profile? Coarse graining a PWM to something like 250 nm resolution results in a measure that is close to only reporting on the GC or AT content, please comment.\n7) In Figure S12, YAP is shown in the multi-color images. Has it been determined if YAP-mCherry signal is not just the TEAD4-eGFP bleed-through signal?\n8) YAP-mCherry is shown not to associate with the Lambda DNA on its own (Figure S13), and at 5 nM (Figure S12), there appears to be no YAP-mCherry signal on the DNA when mixed with TEAD4-GFP. This is not consistent with the proposed assembly model put forth in Figure 7 for cofactor modulation. Additionally, then how would an augmentation (as is described for cofactor affected TEAD4 specificity) of the sequence specificity be substantiated? What is the meaning of improved sequence specificity? Is this being used as a statistical term?\n9) There appear to be no clear error bars or statistics present on the PCC values. Are these available?\n10) For Figure 5D, the residence times are reported for what concentration of TEAD4? Since concentration is an experimental variable, did you choose a single TEAD4 concentration for the residence time of TEAD4 analysis when mixed with co-factors?\n11) Similarly to Figure S12, a figure that shows the 2TDU channel in mixed experiments would be very important for understanding how it may interact with or affect TEAD4, and perhaps even differently than YAP. Showing both channels may be necessary for comprehending the phenomena described, that the added proteins are somehow having an effect on the characteristics of TEAD4 binding, and modifying its behavior from the TEAD4 alone case.\n12) The purpose of using YAP S94A mutant is not clearly explained.\n13) For mixing experiments with sequential addition of YAP-mCherry, the DNA setup is moving from a channel containing TEAD4 and 2TDU (domains of VGLL4) to a channel only containing YAP-mCherry. YAP-mCherry signal and 2TDU signals are extracted from the data. When are the 2TDU signals extracted? This returns to the previous question about the protocol for determining time points for signal quantification. Additionally, are the effects of off-binding of TEAD4 and 2TDU, as a result of being introduced into a new buffer that no longer contain these proteins, considered? Why is the experiment not performed into a buffer that contains the previous proteins at their same concentrations, only with the addition of YAP? Also, TEAD4 profiles are no longer shown in the multi-color image data – is this for a reason, and can these be included, since this system requires this protein for the phenomena observed?\n14) From Figures 6D and 6E, as well as Figure 7, it is concluded that the ratio of 2TDU to TEAD4 is important for YAP recruitment. In Figure 6D, there is colocalized signal of 2TDU and YAP but not in Figure 6E, and a ratio of components argument is made in the text and Figure 7. However, this is not addressed clearly from the data itself. This is directly quantifiable from the data, because actual photon counts (not normalized) should be available, and then the ratios within protein clusters can be directly assessed. If this analysis is done, is it consistent with the subsequent model in Figure 7 and the claim that component ratio is either enhancing or preventing multi-species clusters?"}, "author_response": "We greatly appreciate the comments and suggestions from the reviewers to improve our manuscript. We have carefully considered each comment and taken comprehensive actions as detailed in the following response. The changes made to the manuscript are highlighted in red. We begin below with a summary of major changes in the main text. This is followed by a detailed, point-by-point response to each comment. Each comment is shown in black (bold) and is followed by our response in blue (not bold).\nSummary of major changes to the manuscript:\n1. To provide a more accurate and interpretable representation of the three-component interactions, we carried out experiments, added TEAD4-eGFP fluorescence images to complement the previously shown co-localization between 2TDU-LD655 and YAP-mCherry at low 2TDU concentrations, and replaced normalized fluorescence values with absolute photon counts. These data have been updated in the new Fig. 6d-e.\n2. To provide structural hypotheses supporting our proposed mechanisms, we included AlphaFold3-predicted structural models of various TEAD4 complexes. These data have been updated in the new Supplementary Fig. 2.\n3. To clarify whether TEAD4 could multimerize in solution, we included kymographs acquired in the protein channel at higher TEAD4 concentrations, showing that TEAD4-eGFP multimerization can occur prior to DNA binding. These data have been updated in the new Supplementary Fig. 8.\n4. To more rigorously distinguish multimeric TEAD4 binding from tightly packed monomeric binding on the 10× MCAT array, we performed an additional unzipping experiment to determine the footprint of monomeric TEAD4 on the MCAT motif, and analyzed the spacing of consecutive force peaks generated by TEAD4-eGFP on the 10× MCAT DNA. These data support that TEAD4 forms multimers on the 10× MCAT DNA and have been updated in the new Supplementary Fig. 13.\n5. To better resolve how cofactor dosage influences TEAD4 multimerization, we added an intermediate cofactor concentration in the experiments examining YAP- and 2TDU-induced TEAD4 multimerization, and quantified fluorescence from individual TEAD4 binding events. These data reveal a concentration-dependent effect of cofactors on TEAD4 multimerization and data have been updated in the new Supplementary Fig. 14c.\n6. To directly visualize how TEAD4 occupancy influences subsequent 2TDU recruitment, we performed experiments and added confocal images showing 2TDU-LD655 binding after TEAD4-bound DNA tethers were transferred into a 2TDU-LD655 channel. These data have been updated in the new Supplementary Fig. 16b.\n7. To provide quantitative support for the proposed stoichiometry-dependent model, we quantified fluorescence intensity ratios within TEAD4-eGFP, YAP-mCherry, and 2TDU-LD655 triple complexes. These data further confirm the conclusion that YAP binding depends on the relative ratio of VGLL4 to TEAD4 and have been updated in the new Supplementary Fig. 19."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a good job addressing the points of the reviewers. Our only remaining comment is that they should provide information on the reliability of the AF3 predictions for structure and for interactions. They just need to show the models colored by the pLDDT including their PTM, iPTM scores and the PAE matrix. Otherwise we believe the paper is ready for publication", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have thoroughly addressed the concerns from me and the other reviewers. I thus recommend publication of the revised manuscript without delay.", "reviewer_4": "The authors have done a laudable job at addressing most remarks that were raised. However, there are still concerns for some comments, which were not fully addressed.\nThe most notable is regarding the definition of multimerization raised in Comment #3. The authors added the intensity from a single eGFP in Figure 3D. However, this does not address the concern of whether the protein is actually undergoing multimerization and if the signal analysis alone is sufficient to demonstrate multimerization.\nThe individual photon counts are, as expected, higher than a single eGFP molecule with the repeated motif (10x MCAT) experiment. However, a comparison should be made to the intensity that could arise from 10 binding events (also taking into consideration the resolution, which the authors state as “≈ 250–300 nm”), i.e., a monolayer saturation without multimerization, which is plausible for the system, since there are 10 times repeat of the motif. However, the data shows the signal is actually on average only approximately 2x the photon count from a single eGFP (Figure 3D).\nTherefore, from the data presented, it is difficult to understand if multimerization is actually taking place. The authors present the argument: “Instead, we mainly inferred multimerization from the observation that TEAD4 molecules slide collectively along the 10×MCAT sites as a single complex (new Supplementary Fig. 11b), supporting that TEAD4 binds as a multimeric assembly rather than independent monomers.” However, this statement directly contradicts the authors’ Supplementary Figure 5: Monomeric TEAD4 diffuses along the DNA.\nA clear demonstration of multimerization is key to the main conclusions of the paper.\nIn response to Comment 2, the authors provide: “…we have added kymographs for 1, 5, and 10 nM TEAD4 in the protein channel, with background subtraction applied. These data more clearly illustrate that TEAD4 multimerization can occur both on the DNA surface and in the bulk solution.” This is a new and interesting point, which can change the understanding of the system in both the TEAD4 only and TEAD4 associating with cofactor experiments. Notably, it complicates the understanding if multimers would be facilitated by the DNA surface or are already greatly present in the protein solution at 10 nM. Did post-protein purification analysis reveal the presence of higher order structure formation (i.e., SDS-PAGE, mass photometery, other analytical methods) and could this be provided? Also, since YAP is stated to “…preferentially associate with multimeric TEAD4 assemblies,” it would be helpful to know if these structures pre-form away from DNA and then attach to DNA as a multimeric complex. For this reason, representative kymographs or movies of experiments that involved TEAD4 plus cofactors would be helpful to better understand the nature of the system.\nLastly, as just a point of clarification, in Figures 6D and 6E, the length of DNA is notably very different for the two different representative experiments shown. However, the authors state that: “In our single-molecule assays, the DNA tether was maintained at a constant distance…”"}, "author_response": "We again greatly appreciate the comments and suggestions from the reviewers to further improve our manuscript. We have carefully considered each comment and taken comprehensive actions as detailed in the following response. The changes made to the manuscript are highlighted in red. We begin below with a summary of major changes in the main text. This is followed by a detailed, point-by-point response to each comment. Each comment is shown in black (bold) and is followed by our response in blue (not bold).\nSummary of major changes to the manuscript:\n1. To further support TEAD4 multimerization in solution, we performed the mass photometry analysis as suggested. These data further strengthened our conclusion and have been updated in the new Supplementary Fig. 8.\n2. To enhance the multimerization signal of TEAD4 at the 10×MCAT sites, we increased the protein concentration. These data have been updated in the new Supplementary Fig. 11.\n3. To show TEAD4 and YAP could pre-form away from DNA and then attach to DNA as a multimeric complex, we provided kymographs showing YAP-mCherry immediately binding to the DNA at the beginning of transferring to the protein channel. These data have been updated in the new Supplementary Fig. 20."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have done a laudable job at addressing the remaining concerns. The paper should be publishable as is."}, "author_response": ""}]

Decision letter