The coordinated regulation of transcriptional networks underpins cellular identity and developmental progression. RNA polymerase II promoter-proximal pausing (Pol II pausing) is a prevalent mechanism by which cells can control and synchronize transcription. Pol II pausing regulates the productive elongation step of transcription at key genes downstream of a variety of signalling pathways, such as FGF and Nodal. Recent advances in our understanding of the Pol II pausing machinery and its role in transcription call for an assessment of these findings within the context of development. In this review, we discuss our current understanding of the molecular basis of Pol II pausing and its function during organismal development. By critically assessing the tools used to study this process we conclude that combining recently developed genomics approaches with refined perturbation systems has the potential to expand our understanding of Pol II pausing mechanistically and functionally in the context of development and beyond. A ranromoters .Over the past few decades, our cumulative understanding of the biochemical basis of transcription has revealed that it does not function as a simple ‘on-off’ switch, but instead involves a sequence of steps that each contribute to the overall transcription rate ,4–6. Broin vitro, for the mid-blastula transition (MBT) in fruit flies, hematopoietic stem cell specification in zebrafish and for early embryo development in mouse [in vitro cell culture models or invertebrate (Drosophila melanogaster) embryos, while relatively little is known about the specific roles of Pol II pausing in mammals.The molecular mechanisms responsible for establishing and preserving Pol II pausing have been extensively studied over the last two decades. Genetic ablation of various components of the Pol II pausing machinery has determined that it is required for cell proliferation in mouse –18. Thesin mouse ,19. Intrin mouse . AdditioIn a mammalian system, an extensive analysis of the Pol II landscape in early pre-implantation mouse development was recently reported, which revealed dynamic pausing at distinct stages between the zygote and early blastocyst stages . Pre-impIn this review, we focus on Pol II pausing from a functional perspective during embryo development. We start by briefly discussing our molecular understanding of Pol II pausing and highlight recent discoveries. We then discuss the established roles for Pol II pausing in different developmental models, with a focus on early mammalian development, and the current limitations and open questions in the field. Finally, we discuss how these limitations could be overcome to gain a deeper understanding of the functional relevance of Pol II pausing during development.. 2. 2.1Promoter-proximal RNA polymerase II pausing is defined as a transcription halt following initiation but prior to elongation ,13. FollFor Pol II to proceed to elongation and transcription of the remaining gene body, the paused complex must be released. Paused Pol II is released by an array of general elongation effectors such as BRD4, TRIM33 and the Mediators which ultimately recruit the positive transcription elongation factor, or P-TEFb ,34–36. T. 2.2D. melanogaster embryos [Drosophila were used to characterize the determinants of, and factors involved in, Pol II pausing [Pol II pausing was initially discovered on heat shock responsive genes in Schneider line 2 (S2) cells derived from  embryos . Paused  embryos ,42. Thus pausing ,44. Rece pausing ,45. Thes pausing ,47, rang pausing ,15,48. TDrosophila Kc167 cells, inhibition of transcriptional elongation using flavopiridol results in all active genes accumulating paused polymerases, while inhibiting initiation revealed a clear difference in the stability of paused complexes at individual gene loci (ranging from minutes to hours) [To interrogate the dynamics of Pol II pausing, acute perturbation of transcription initiation or elongation have been carried out using chemical inhibitors such as triptolide and flavopiridol, respectively –51. Theso hours) ,51. Theso hours) . Overallo hours) .. 2.3et al. and Shao et al. showed that Pol II pausing inhibits new PIC assembly [−1, while paused Pol II can be stable for 1–10 min on average, and as long as one hour at some promoters [While Pol II pausing has been implicated as a key rate-limiting step in transcription, only recently has clear molecular and causal evidence for such a role been demonstrated, highlighted by two key findings. First, by using a combination of improved ChIP- and nascent RNA-seq approaches to locate RNA Pol II on the genome and determine nascent transcriptional output, Gressel assembly ,51. Cryoassembly . Second,assembly . SpecifiOf note, the stability, dynamics and turnover of paused Pol II at protein-coding genes has been studied by several groups, albeit with some discrepancy regarding the exact time estimates ,51,53–55a). However, since complete depletion of Pol II pausing components can take days to achieve (as in these studies), it is challenging to delineate gene loci directly affected by Pol II pausing from ones affected by secondary or compounding effects of these perturbations and the time scale of analyses. Still, these conclusions provide new and enticing avenues for investigating the endpoint consequences of perturbing pausing on the transcriptional activity of genes, and the overall transcriptional state of cells, even though they are limited by the tools employed, and thus lack direct experimental evidence.It remains unclear how Pol II pausing can directly influence overall transcriptional output from a particular locus. Several loss-of-function experiments of pausing components, such as the NELF complex, revealed that this can result in either up- or downregulated expression of highly paused genes in a variety of model systems ,56,57. Tfigure 2. 2.4b). This interaction is abrogated when additional bases are added between the PIC site and pausing site [in vitro, and a degron-based approach to test the hypothesis in cells. However, validation in cells is complicated by changes in transcriptional dynamics upon TFIID degradation that may be intrinsic to PIC assembly and pause-release rather than pausing. Therefore, the extent to which Pol II pausing might regulate new PIC assembly remains unclear, and conversely, feedback from the PIC itself onto the pausing machinery is yet to be evaluated. Nevertheless, these data strongly argue that while Pol II pausing may inhibit new Pol II initiation, it does not completely inhibit binding of other general PIC transcription factors which can keep a promoter ‘marked’ for activation.When considering a hypothetical gene, the Pol II pausing machinery lies near the PIC just upstream, with regulatory chromatin structure on the gene body downstream. Thus, it is fair to assume that these complexes might interact with each other. Upstream, with respect to the PIC, recent work suggests that the general transcription factor IID (TFIID) plays an active role in maintaining Pol II pausing via a direct or indirect interaction with the paused polymerase complex given their physical proximity b. This iing site ,59. Thisb). A recent study suggests that other unidentified histone deacetylases may have a similar impact to SIRT6 on Pol II pausing [It is also plausible that the pausing complex mediates chromatin accessibility and shapes the histone modification landscape around the TSS. The histone H3 lysine 4 trimethylation (H3K4me3) and lysine 9 acetylation (H3K9ac) modifications mark active promoters . H3K4me3figure 2 pausing . These s. 3Drosophila, zebrafish and mouse [Faithful development of organisms requires coordinated and precise regulation of gene expression. Rapid responses to stimuli—both, extrinsic and intrinsic—dictate specific spatio-temporal gene expression patterns . The resnd mouse –18. In t. 3.1Drosophila has been the most extensively studied model for characterizing Pol II pausing [cis-regulatory elements that identify specific sequences. On the other hand, adult tissue-specific genes display Pol II pausing to a lesser extent, and tend to be driven by TATA box-containing promoters. The precise purpose of having these developmental genes paused prior to productive elongation has only been speculated. Since Pol II pausing is enriched at developmental and rapid response genes, it might aid in speed of activation, synchronized and co-regulated expression and the insulation of promoters from chromatin compaction [ pausing . NELF is pausing . Subsequ pausing ,69. ChIPmpaction ,29,70.. 3.2Drosophila. This is due to the more complex nature of mammalian development and limited accessibility to large amounts of embryonic material. Nevertheless, loss-of-function studies in mouse embryos have revealed an essential role for NELF during early development. Nelf mutant embryos fail to progress beyond mid-gestation (approx. embryonic day (E) 8.0–9.0) [In general, our understanding of Pol II pausing in mammalian systems lags that in 8.0–9.0) ,18, perhin vitro [Drosophila is enriched at developmental genes, mES cells do not show a specific enrichment of Pol II pausing at developmental genes [mES cells can recapitulate many aspects of embryonic development, and thus have been used as a tractable and scalable model to study key developmental processes in vitro . Pol II in vitro . Of noteal genes . Human Eal genes .Nelfb, a subunit of the NELF complex, revealed that while developmental and stem cell identity genes are unaffected, the genes which are mis-regulated coordinate responses to extracellular signals. By contrast to wild-type mES cells, Nelfb mutant cells are resistant to spontaneous differentiation. On a molecular level, transcriptional targets of FGF and WNT signalling pathways are affected, resulting in impaired downstream responses to these signalling cues [in vitro? Can the defects observed in mES cells shed light on the cause of lethality in embryos? As mentioned previously, interpretation of the results from studies in mES cells need to be de-coupled from potentially confounding secondary effects, such as cell cycle defects, which have been reported in these studies.Manipulating Pol II pausing in mES cells via genetic deletion of ing cues ,18,25. Ting cues –75. TherOther studies have also examined Pol II pausing in the context of regeneration via tissue specific NELF knockout in mice ,77. Mech. 3.3Drosophila to humans, genome-wide studies have highlighted that immediate-early release genes that respond to signalling pathways tend to be enriched for paused Pol II complexes [From the initial identification of the evolutionarily conserved Pol II pausing at heat-shock responsive genes from omplexes ,78–80. Tomplexes ,18,79–84omplexes . This coα) directly interacts with NELFB and p-TEFB, and might mediate their recruitment to Pol II depending on signalling state (i.e. active or inactive) [α recruits P-TEFb to the MYB gene to drive transcription beyond the regulatory SL-dT region—a Pol II pausing site approximately 1.7 kb downstream of the TSS [a).The role of Pol II pausing in mediating specific responses to cellular signalling was demonstrated prior to its proposed function in the context of development. In addition to the previously discussed signalling pathways, retinoic acid and oestrogen signalling can also modulate transcription specifically via control of Pol II pausing in mammalian cells, while disruption of this control is implicated in disease states such as cancer –85. Oestnactive) ,82. In mnactive) ,84. In sAn alternative mechanism by which signalling may regulate Pol II pausing and pause-release is by controlling the activity of specific transcription factors. c-MYC, a pioneering transcription factor implicated in development and disease and controlled directly via the LIF/JAK/STAT signalling pathway, can regulate downstream gene expression primarily through pause-release and promoting active elongation in mES cells ,86. c-MY. 3.4in vivo could not be examined due to limited biological material. By leveraging a transposase-coupled antibody assay with low input (approx. 500 cells), Liu et al. were able to map Pol II occupancy in pre-implantation mouse embryos [Drosophila embryos during the pre-MBT stage, which also coincides with ZGA [Until recently, the genomic landscape of Pol II binding during early mammalian development  embryos . They prwith ZGA ,69. Notawith ZGA .Nelfb in early post-implantation development and mES cell differentiation are consistent with a role in the re-establishment of pervasive Pol II pausing at active and inactive promoters after the emergence of pluripotency in the blastocyst (i.e. specification of the EPI lineage ∼E3.5) [Altogether, these studies suggest that Pol II pausing may be required at distinct times in early mammalian development including two key stages—ZGA and EPI maturation and differentiation. The functional role of Pol II pausing during mammalian ZGA has not yet been assessed. By contrast, the requirement for e ∼E3.5) ,18.Nanog and Klf2 in the embryo as well as in mES cells, and genomic priming of formative markers, such as Pou3f1, Lef1 and Fgf5, for efficient progression away from the naive state [in vitro by alleviating TCF3 (Tcf7l1) repression of naive pluripotent markers, its inhibition is required to maintain the primed state of pluripotency [Once pluripotency is established in the embryo, it is quickly disassembled as development proceeds. The pluripotency ‘continuum’ transitions from a ‘naive’ state in the blastocyst toward a ‘formative’ state in the newly implanted embryo before acquiring a ‘primed’ state as cells prepare for germ layer differentiation during gastrulation ,89. Durive state ,100–103.ipotency ,104–109.ipotency ,111. TimNelfb mutant embryos results in embryonic lethality around the time of gastrulation and mutant mES cells display an attenuated response to differentiation cues, thus supporting such a proposed role for Pol II pausing during development [Considering that the establishment of pluripotency in the mouse embryo through to the initiation of gastrulation encompasses just three days of development, the reorganization of transcriptional networks and careful coordination of multiple signalling inputs in space and time must require a fine-tuned system of gene activation and repression. Since Pol II pausing has been reported in other mammalian and invertebrate systems as mediating rapid and coordinated transcriptional responses to signalling inputs, it may also function in a similar capacity during pluripotency progression and gastrulation. Loss of Pol II pausing in elopment ,18,20. Melopment ,103.. 4. 4.1de novo at many rapidly induced FGF/ERK targets in mES cells upon signal induction [There is evidence that Pol II pausing facilitates rapid induction of gene transcription, leading to the hypothesis that such genes might be primarily regulated at the level of pause-release with an inducing factor—a transcription factor or signalling effector—responsible for recruiting factors to release paused Pol II ,29. Thisnduction . These fERK activity in the mouse blastocyst is also known to be dynamic with subtle but specific differences between cell lineages driving the specification and later priming of the pluripotent EPI and mES cells ,118. The. 4.2In mammalian cells, synchronous induction of genes may be of particular importance for developmental signalling pathways that have several negative feedback loops, such as FGF/ERK and Nodal/Smad2 signalling, which can be induced rapidly to maintain specific levels of pathway activity and induce tissue morphogenesis ,119,120.in vitro and facilitate EPI maturation in vivo [Synchronous gene induction not only requires paused transcription, but also strict regulation of Pol II pausing to prevent aberrant expression. Thus, Pol II pausing serves as a second ‘checkpoint’, along with Pol II recruitment, in a two-step control system to ensure robust induction without low or basal transcriptional activity . The fir in vivo ,123,124. in vivo ,125.Fig. 4.3b).Pol II pausing may also play a passive role in development simply as a function of its physical presence and kinetic stability relative to other components of the transcriptional machinery. In the mouse blastocyst, as EPI and PrE cells are specified and subsequently mature, their overall chromatin structures transition to a more compact, inactive state ,127. It figure 3Drosophila S2 and mES cells [Recently, widespread Pol II pausing was also detected at actively transcribed enhancers in ES cells . It is yES cells –130, havES cells  (figure Drosophila, and remain associated with paused polymerases at target gene promoters [While Pol II pausing may keep promoters and enhancers protected from nucleosomes and accumulation of repressive histone marks, the molecular mechanisms for how this is accomplished have yet to be characterized. It is possible that the pausing complex prevents promoters from being closed between ‘transcriptional bursts' when Pol II complexes are not actively transcribing. These bursts are a well-documented phenomenon of mammalian transcription, occurring minutes to hours apart—a time scale that allows for some chromatin modifications and rearrangements ,131,132.romoters . Alternaromoters ,137.Addressing these possible roles for Pol II pausing in the context of mammalian development would improve our understanding of transcriptional regulation and successful establishment of resulting cellular states. Moreover, it may help explain how cells translate extrinsic or intrinsic stimuli for rapid and coordinated transcriptional responses driving key differentiation and morphogenetic processes that constitute developmental progression.. 5It is important to discuss the experimental approaches that have been used to make the conclusions discussed so far. Studying global transcription dynamics has been facilitated by advances in nascent RNA sequencing techniques allowing detailed identification of sites of Pol II pausing and kinetics ,13. By cMore importantly, transcription is a process that proceeds on a time scale of seconds to minutes ,51. To tDrosophila to mice, with increasing evidence that it provides a crucial link between cellular signalling inputs and transcriptional outputs during a variety of cellular state transition events. With recent high-resolution analyses of Pol II pausing in early mammalian development and tools for rapid perturbations, the door is open to dissect the specific functions of Pol II pausing in mammalian development.RNA Pol II pausing has emerged as a widespread phenomenon in metazoans, and a potential node for gene regulation in mammalian systems. There is a growing list of molecular interactions and functions of the pausing complex, yet it has been challenging to attribute cellular phenotypes to molecular perturbations of Pol II pausing. In particular, Pol II pausing is critical to organismal development from


The YAP and TAZ paralogs are transcriptional co-activators recruited to target sites by TEAD proteins. Here, we show that YAP and TAZ are also recruited by JUNB (a member of the AP-1 family) and STAT3, key transcription factors that mediate an epigenetic switch linking inflammation to cellular transformation. YAP and TAZ directly interact with JUNB and STAT3 via a WW domain important for transformation, and they stimulate transcriptional activation by AP-1 proteins. JUNB, STAT3, and TEAD co-localize at virtually all YAP/TAZ target sites, yet many target sites only contain individual AP-1, TEAD, or STAT3 motifs. This observation and differences in relative crosslinking efficiencies of JUNB, TEAD, and STAT3 at YAP/TAZ target sites suggest that YAP/TAZ is recruited by different forms of an AP-1/STAT3/TEAD complex depending on the recruiting motif. The different classes of YAP/TAZ target sites are associated with largely non-overlapping genes with distinct functions. A small minority of target sites are YAP- or TAZ-specific, and they are associated with different sequence motifs and gene classes from shared YAP/TAZ target sites. Genes containing either the AP-1 or TEAD class of YAP/TAZ sites are associated with poor survival of breast cancer patients with the triple-negative form of the disease. The Hippo signal transduction pathway plays critical roles in development, homeostasis, and tumor progression . InternaDeregulation of YAP/TAZ activity is frequently observed in various human cancers, contributing to cancer initiation, progression, and metastasis . YAP/TAZIn addition to directly interacting with the TEAD family of DNA-binding proteins, YAP/TAZ can function through other mechanisms. Via its interaction with TEAD proteins, YAP/TAZ can synergize with JUN/FOS at composite regulatory elements containing both TEAD and AP-1 motifs . YAP-TEAIn previous work, we described an epigenetic switch that transforms breast cells via an inflammatory regulatory network controlled by the joint action of NF-κB, STAT3, and AP-1 transcription factors . Here, wGiven the importance of YAP and TAZ in many types of human cancer, we examined their roles in breast cellular transformation by using CRISPR to knock out these genes in the context of our Src-inducible model . Treatment of these non-transformed cells with tamoxifen activates v-Src and triggers an epigenetic switch to the transformed state. Knockout of YAP or TAZ or both  reduces An inflammatory regulatory network mediated by the joint action of NF-κB, STAT3, and AP-1 factors at common target sites is critical for transformation in our inducible model, and this network is involved in many human cancers . Using sEscherichia coli  that mediate interactions with a variety of other proteins. To map the regions of YAP and TAZ required for interacting with STAT3 and JUNB, we performed co-IP experiments in cells co-expressing YAP or TAZ derivatives lacking one or both WW domains along with FLAG-tagged STAT3 or JUNB . The WW1When overexpressed in parental MCF-10A cells , wild-type YAP or TAZ increase the level of transformation, whereas derivatives lacking YAP-WW1 or TAZ-WW do not . Like thUsing protein-specific antibodies, we performed ChIP-seq to identify the genomic target sites of YAP, TAZ, STAT3, JUNB, and TEAD in both non-transformed and transformed cells. In general, there is excellent agreement in genome-wide signal profiles between pairs of biological replicates for each factor , and theAlthough nearly all target sites are bound by both YAP and TAZ, ~ 575 target sites (~2% of YAP/TAZ sites) appear specific for either YAP or TAZ . To testSeveral characteristics of these YAP- and TAZ-specific sites suggest that they serve biologically distinct functions from shared sites. First, >80% of YAP- and TAZ-specific sites are located within 150 base pairs upstream of a GENCODE-annotated transcription start site (TSS), but <25% of shared YAP/TAZ sites are so localized . Second,We compared YAP/TAZ binding data with that of STAT3, JUNB, and TEAD proteins . ConsistWe further investigated protein co-occupancy by performing pairwise analysis of ChIP-seq peak summits in transformed cells . Such anIL-6 enhancer, sequential ChIP in either order yields increased fold-enrichments of YAP  of JUNB and either YAP or TAZ on a transfected plasmid-borne locus containing six AP-1 motif sites shows increased fold-enrichment in transformed cells, while this does not occur on a control plasmid locus lacking the AP-1 sites  for AP-1, TEAD, and STAT3 motifs located ±150 bp from every YAP/TAZ peak summit. Of particular interest, we identified AP-1, TEAD, and (to a lesser extent) STAT3 classes of YAP/TAZ sites with high FIMO scores for the defining motif and low scores for the other two motifs . In accoThe observation that JUNB, TEAD, and STAT3 co-associate with distinct classes YAP/TAZ sites suggests that different forms of an AP-1/TEAD/STAT3 complex can recruit YAP/TAZ. To examine this possibility, we measured the relative crosslinking efficiencies of these DNA-binding proteins at different classes of YAP/TAZ sites. Strikingly, the JUNB:TEAD crosslinking ratio is strongly correlated with the quality of the AP-1 motif, whereas it is negatively correlated with the quality of the TEAD motif . Such diUnexpectedly, the motif recognized by the CEBP family of transcription factors occurs in 20% of YAP/TAZ sites with an enrichment over control sites that is second-most behind that of TEAD motifs . CEBP moAnalysis of ~450 TAZ-specific sites  indicateTo provide independent support of our results, we performed ChIP-seq in a triple-negative breast cancer cell line (MDA-MB-231). As is the case in ER-Src cells, binding profiles for YAP, TAZ, STAT3, JUNB, and TEAD are extremely similar , and theInterestingly, there are differences between the two cell lines with respect to how YAP/TAZ is recruited to some target loci. At YAP/TAZ target sites, the enrichment of CEBP motifs in ER-Src cells is not observed in MDA-MB-231 cells . ConversWe determined which genes are associated with the different classes of YAP/TAZ target sites in ER-Src cells . For simTo identify genes regulated by YAP, TAZ, JUNB, STAT3, or TEAD proteins, we individually depleted these factors by siRNA-mediated knockdown in tamoxifen-treated ER-Src cells. RNA-seq analysis identifies between 1000 and 4000 differentially expressed genes for each condition when compared with a control siRNA. Roughly equal number of genes show increased or decreased expression with the directionality of differential expression nearly always preserved among factors . In accoThe TCGA Invasive Breast Carcinoma Dataset includes 1108 breast cancer samples from 1101 patients with follow-up information, of which 116 are triple-negative breast cancers (TNBC). To determine the clinical significance of YAP/TAZ target genes in breast cancer samples, we performed Kaplan–Meier survival analysis on gene sets based on motifs at the YAP/TAZ site within 2 kb of the transcription start site of all YAP/TAZ targets. For each gene set, we computed a gene signature score (GSS) for each patient and then divided patients into low (GSS < 0) and high (GSS > 0) gene signature expression (low and high risk). Kaplan–Meier analysis was performed on the entire set of patients as well as patient subsets with luminal A, luminal B, HER2+, and TNBC cancer types.Notably, high expression of the gene subset with YAP/TAZ sites having only AP-1 motifs is associated with significantly shorter survival in TNBC patients ; p=0.023As YAP/TAZ target sites with both AP-1 and TEAD motifs are strongly associated with poor survival of TBNC patients, we asked whether sites with multiple AP-1 or TEAD motifs have a larger impact on survival than sites with just one motif. We defined sets of genes with proximal YAP/TAZ sites having exactly one (120 genes) or two or more (167 genes) AP-1 motifs . We defined the same sets for TEAD motifs . YAP/TAZ target sites with one or multiple AP-1 motif sub-signatures are associated with significant survival differences, with sites that contain multiple motifs having a slightly higher significance . Fourth, many YAP/TAZ target sites coincide with AP-1 and, to a lesser extent, STAT3 motifs. Indeed, the frequency of AP-1 motifs among YAP/TAZ target sites is comparable with that of TEAD motifs. Fifth, sequential ChIP experiments directly show that YAP/TAZ co-occupy target sites with JUNB and STAT3.Here, we provide multiple lines of evidence, demonstrating that YAP and TAZ are also transcriptional co-activators of STAT3 and AP-1 proteins. First, YAP and TAZ co-immunoprecipitate with JUNB and STAT3 in nuclear extracts. Second, all direct pairwise interactions between YAP or TAZ with JUNB or STAT3 are observed with proteins expressed in The current model is that the YAP/TAZ co-activators are recruited by TEAD proteins that act together with AP-1 factors at genomic regions with composite sites containing AP-1 and TEAD sequence motifs . ConsistBased on our results, we propose a new model in which YAP/TAZ is recruited to target genomic regions by a complex of AP-1, STAT3, and TEAD proteins . The priThis model is based on three lines of evidence. First, at YAP/TAZ target sites, pairwise peak analysis shows that the distances between AP-1, STAT3, TEAD, and YAP/TAZ are comparable to the distances between biological replicates of an individual protein. This indicates that the binding locations of all three DNA-binding proteins are very close to each other, and likely coincide, at the vast majority of genomic targets. This co-localization also suggests direct recruitment of YAP/TAZ to these target sites by one or more of the DNA-binding proteins.Second, all three DNA-binding proteins can associate with genomic regions that have a single AP-1, STAT3, or TEAD motif, and YAP/TAZ binding peaks coincide with individual motifs. Such YAP/TAZ sites with only a single motif strongly suggest the existence of an AP-1/STAT3/TEAD complex. ChIP-seq experiments do not formally exclude the possibility that AP-1, STAT3, and TEAD occupy target regions at different times. However, this seems very unlikely at sites with only a single motif because it is hard to imagine how the non-cognate proteins would recognize the motif in the absence of the cognate protein. In addition, these considerations indicate that composite motifs are not necessary for YAP/TAZ recruitment by the AP-1/STAT3/TEAD complex. For targets with composite motifs, it is unclear whether both motifs are necessary for YAP/TAZ recruitment or whether separate AP-1/STAT3/TEAD complexes form on the individual motifs. Although not mutually exclusive, we favor the latter possibility because the distance between TEAD and AP-1 motifs is quite variable at YAP/TAZ target sites with these composite motifs.Third, the relative crosslinking efficiencies of the DNA-binding proteins differ at the distinct classes of YAP/TAZ sites, strongly suggesting different forms of the AP-1/STAT3/TEAD complex. In contrast, monolithic complexes  show invariant crosslinking ratios among factors at all target sites . At an iStructural details of the putative AP-1/STAT3/TEAD complex and the mechanism of YAP/TAZ recruitment remain to be elucidated. It is unknown whether AP-1, STAT3, and TEAD interact directly with each other, whether YAP/TAZ helps stabilize the complex, and whether other proteins are involved. The existence of other proteins in the complex seems possible given that some YAP/TAZ target sites lack any of these motifs. It is likely that STAT3 is not an obligate component of the complex because JUNB, TEAD, and YAP/TAZ occupancies are similar in transformed and non-transformed ER-Src cells, which have different levels of STAT3 occupancy. Finally, JUNB, STAT3, and TEAD can directly interact with YAP/TAZ, but the relative and possible synergistic contributions of the DNA-binding proteins to YAP/TAZ recruitment are unknown. Understanding these structural details will require atomic resolution of the complex on the different classes of target sites.Genetic experiments indicate that YAP and TAZ have distinct and overlapping cellular functions , but theA significant minority of YAP/TAZ target sites are not associated with AP-1, STAT3, or TEAD motifs, suggesting that other transcription factors can help recruit YAP/TAZ. CEBP motifs are enriched and frequently observed at YAP/TAZ target sites in ER-Src cells, but not in MDA-MB-231 cells. Conversely, RUNX3 motifs are enriched at YAP/TAZ target sites in MDA-MB-231 cells, but not in ER-Src cells. Such cell-line specificity could be due to differences in the amount or activity of the recruiting protein(s) that recognize the motif and/or differences in auxiliary proteins that are important for recruitment to these motifs.YAP/TAZ target genes associated with AP-1 or STAT3 motifs are functionally distinct, but both are associated with poor survival of patients with the triple-negative form of breast cancer.The current view is that the biological functions of YAP/TAZ are mediated primarily through their interaction and recruitment by TEAD proteins . HoweverMCF-10A-ER-Src cells  were gro4 cells in culture medium were mixed with 0.4% low-melting-point agarose  at 37°C and seeded on top of 1% agarose standing layer in 12-well dishes. Colony density was measured 2–3 weeks after seeding with images captured by a digital camera . For the GILA assay, 2000 cells were seeded into ultra-low attachment surface 96-well plate . Five days after seeding, sphere cells growing in the low attachment plates were quantitated by CellTiter-Glo luminescent cell viability assay  using a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices). Cells growing in regular culture (high attachment) dishes were stained using crystal violet, and cell density was measured using Fiji Image J’s (version 1.52b) measurement function.The transformation capacity was measured by growth in low attachment conditions (GILA)  or in soAAACTCTCATCCACACTGTTCAGGC and CACCGCCTGAACAGTGTGGATGAGA; TAZ exon 3 – AAACCCCGACGAGTCGGTGCTGGAC and CACCGTCCAGCACCGACTCGTCGGG. CRISPR lentiviral plasmids and VSV-G, GP, and REV plasmids were transfected into 293T cells to produce CRISPR lentivirus. CRISPR lentiviruses infected ER-Src cells for 1 day, and cells were selected with 10 μg/ml blasticidin  for additional 3 days.CRISPR sgRNAs, designed with previously described algorithms , were clOligo siRNAs were purchased from Dharmacon (siGENOME SMART pool)  and wereCell fractionation protocols were like the protocols described before  with somCo-immunoprecipitations were performed as described previously . BrieflyRNA was extracted using mRNeasy Mini Kit . One microgram RNA was converted to cDNA using SuperScript III Reverse Transcriptase . qPCR was running using a 7500 Fast Real-time PCR system (Applied Biosystems). qPCR primer sequences can be found in IL-6 secretion was measured using human IL-6 immunoassay kit  as per manufacturer’s instructions. Optical density was determined using SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices).The pGL-AP-1 plasmid containing six consensus AP-1 binding sites was co-transfected with the pRL-CMV plasmid (Promega) into cells using TransIT 2020 transfection reagent . A pRL-CMV plasmid expressed Renilla protein and was used as an internal transfection control. Twenty-four hours after transfection, cells were split and treated with tamoxifen for 24 hr to induce transformation. After 3 days, firefly and Renilla luciferase activities were determined by Dual-luciferase Reporter Assay kit . To evaluate NF-κB activity, pGL4.32 (luc2P/NF-κB-RE/Hygro)  and pRL-CMV were co-transfected into cells and the same protocol followed.E. coli and dialyzed in a neutral buffer . The direct interactions among these recombinant proteins were examined in vitro using a similar co-IP procedure as mentioned above.To produce recombinant proteins, YAP, TAZ, STAT3, and JUNB were cloned into pET30 plasmid. Recombinant proteins were produced in BL21 Cells were dual crosslinking with a mixture of 2 mM each of ethylene glycol bis (succinimidyl succinate) and disuccinimidyl glutarate and 1% formaldehyde. Chromatin was digested with 60 units MNase  at 37°C for 10 min and then sonicated using Branson Microtip Sonifier 450 (4 × 15 s at output 4.5 and duty cycle 60%) to the sizes mostly between 150 and 500 bp. Fifty microgram chromatin, antibodies for transcription factors  using Bowtie2 . For posDR-2.0.4  with --sWe performed TF occupancy analysis using their IDR peaks. We used the MACS2 pileup command to generate signal profiles which were smoothed using a 10 base pair Gaussian kernel to reduce noise. We then identified summits as the positions within a peak with the highest signal value. We adjusted IDR peaks to the summits ± 150 regions to avoid the potential bias due to varied ChIP-seq peak length distribution between transcription factors. Bedtools-2.29.2 was used to identify overlapping regions and calculate distance between peak summits.We downloaded DNase hypersensitivity sites (DHSs) from the ENCODE Portal  for ER-Shttps://github.com/krews-community/factorbook-meme  or a TEAD dimer motif meeting the 10–4 threshold. For motif enrichment analysis, we downloaded the complete set of transcription factor motifs in the HOCOMOCO and JASPAR databases  with the ‘biological process’ category . 0.4 μg total RNA was used for RNA-seq library preparation. RNA-seq libraries were generated using TruSeq Ribo Profile Mammalian Kit . RNA-seq libraries were and sequenced at Bauer Core Facility using Hiseq 2000. For analysis, we trimmed adapter sequences, ambiguous ‘N’ nucleotides (the ratio of ‘N’ > 5%), and low-quality tags . Clean reads were aligned against the GENCODE v30 reference transcriptome  using SToutFilterMultimapNmax 20--alignSJoverhangMin 8--alignSJDBoverhangMin 1--outFilterMismatchNmax 999--outFilterMismatchNoverReadLmax 0.04--alignIntronMin 20--alignIntronMax 1000000--alignMatesGapMax 1000000--sjdbScore 1--estimate-rspd --calc-ci’. Differential expression analysis was performed with DESeq2  using RSEM  with theh DESeq2  with defTo assess whether differential genes were significantly more likely to be directly regulated by the transcription factors of interest, we divided differential genes into two classes: TF target genes, having an IDR peak for a given factor within 2 kb of any of the gene’s transcription start sites based on GENCODE v30 annotations, and non-TF target genes, not having a peak within that distance of any TSS. We compared each set of differential genes against a control set of the same number of protein coding genes randomly selected from the GENCODE annotations. A chi-square p-value was computed for the respective fractions of target and non-target genes.https://www.cbioportal.org. These samples were sub-divided into Luminal A, Luminal B, HER2+, and TNBC groups according to ER/PR/HER2 status based on immunohistochemistry (i-μi)/σi, where xi is the expression of i gene in patient samples; μi is mean of i gene in all patient samples; σi is standard deviation. The low-expression (low-risk) group was defined as having GSS < 0, while the high-expression (high-risk) group was defined as having GSS ≥ 0 . YAP/TAZ/JUNB and YAP/TAZ/TEAD gene signatures were defined as follows. Motif-based signatures were defined by (1) identifying all genes targeted by both YAP and TAZ in tamoxifen-treated ER-Src cells using the above criteria for ChIP-seq, then (2) filtering these to contain only genes targeted by YAP/TAZ sites containing a AP-1 motif site but not a TEAD motif site, a TEAD motif site but not a AP-1 motif site, or both a TEAD and a AP-1 motif site, using the 10All sequencing data were deposited on National Cancer for Biotechnology Information Gene Expression Omnibus (GEO). GSE166943 is the accession number for all the data, with GSE166941 being the subset for the ChIP-seq data and GSE166942 for the RNA-seq data. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work shows that effectors in the HIPPO signaling cascade function as co-activators that are recruited by transcriptional activators. Furthermore, these HIPPO signaling effectors play a role in transformation of breast epithelial cells in culture.Decision letter after peer review:eLife. Your article is being evaluated by 3 peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor.Thank you for sending your article entitled "YAP and TAZ are transcriptional co-activators of AP-1 proteins and STAT3 during breast cellular transformation" for peer review at As you will learn from the enclosed reviews and recommendations for the authors, the enthusiasm for the article is dampened by a number of concerns in terms of novelty for the field, preliminary basis of some of the findings, and some shortcomings in experimental design and analysis.Reviewer #1:The authors have used an MCF10A-based inducible Src transformation model to study the contribution of the YAP and TAZ transcriptional coactivators to cell transformation. YAP and TAZ are transcriptional coactivators and terminal effectors of the Hippo pathway. They don't possess direct DNA binding activity, and are generally believed to be recruited to specific genomic DNA sites through "piggybacking" on TEAD family DNA binding proteins. The authors now report that YAP and TAZ can be recruited to chromatin through direct interactions with AP-1 proteins  and STAT3. They further provide evidence that these interactions are important for cell transformation, and may be conducive to worse patient outcome in triple negative breast cancer (TNBC). They also show that while YAP and TAZ share many binding sites on the genome, each of them has also some "private" sites, associated with distinct classes of genes.Strengths:The findings are interesting and shed new light on transcription regulation by YAP and TAZ. In particular, they provide evidence that – at least in the setting of the studied cell transformation model – the reported interactions may be more impactful than the canonical interaction of YAP/TAZ with TEAD family members. The experiments are well performed and very clearly described.Weaknesses:This study has no major weaknesses. Yet, there is room for improving it. Some suggestions are listed below.1.The data in Figure 6B raise the interesting possibility that CEBP family proteins may serve to recruit YAP/TAZ to a subset of binding sites. This is a novel and potentially valuable finding. Regrettably, the authors do not pursue it further by suitable experimental work. The authors show later that YAP/TAZ are not recruited to CEBP motifs in MDA-MB-231 cells; perhaps this putative interaction is therefore deemed by the authors irrelevant for TNBC and therefore less worthy of pursuing? It should be noted, however, that MDA-MB-231 is a mesenchymal TNBC cell line, and as such it is not representative of the majority of TNBC cases. This reviewer feels that the lack of follow-up on the CEBP lead is a missed opportunity, and such follow-up will make the paper stronger and more innovative. Do YAP/TAZ interact directly with CEBP family members? And is their binding to the indicated sites abrogated by depletion of such CEBP proteins?2. The data presented in this study support a model where YAP and TAZ are recruited to AP-1 sites independently of TEAD. However, several earlier studies report that YAP/TAZ transcriptional interaction with AP-1 does occur in concert with TEAD . This should be discussed more thoroughly in the Discussion.3. In view of published work by the Piccolo group showing the prominence of YAP/TAZ binding to enhancers, the authors may wish to compare the patterns of YAP/TAZ binding in concert with AP-1, STAT3 and CEBP between TSS-proximal (promoter) sites and distal enhancer sites.4. The authors may wish to elaborate in more depth and detail on the integration of the chromatin binding site analyses and the functional implications of the transcriptional output of YAP/TAZ/JUNB vs YAP/TAZ/STAT3 vs YAP/TAZ/TEAD.5.Please define the workflow of experiments in greater detail. For instance, in Figure 2A, how long before TAM induction of SRC was siRNA knockdown performed? Also, please define "standard conditions of high attachment". Similarly, figure legends should be more technically informative.6. Figure 3A. According to Figure 2A, STAT3 protein levels do not increase in response to SRC induction. However, "summing up" the protein levels in "E" vs "T", it does look like STAT3 levels increase. Total lysate (input) should be presented. In either case, it would be advantageous to see subcellular localization also by immunofluorescence.7. Was this overexpression and Co-IP experiment performed in MCF10A-ER-SRC cells induced by TAM? If yes, this should be clearly stated.8. Line 130. "the WW domain of TAZ [is] critical for the interaction with STAT3 and JUNB" Actually, for the Co-IP experiments presented in Figure 3B and 3D, it looks like TAZ interacts only weakly (and certainly more weakly than YAP) with JUNB and STAT3. Is this the case? Is this differential YAP/TAZ behavior represented in the ChIP data? .9. Figure 5D and E. Is there no augmented binding of YAP/TAZ/JUNB or STAT3 on the IL6 enhancer following TAM treatment?10. Figure 6A and line 231. It will be helpful to mention that "JUNB" in this figure is synonymous with AP-1 motif.11. The difference and meaning of Figure 6—figure supplement 4B and 4C is not explained well enough.12. Figure 7C is not mentioned in the text. Anyway, what does it add?13. Figure 7E-G. To better assess the impact of YAP/TAZ on STAT3/JUNB function and their relationship to TNBC survival, Kaplan Meier plots should be generated for STAT3/JUNB alone vs STAT3/JUNB/YAP/TAZ.14. The authors report that in addition to many target sites shared between YAP and TAZ, there also exist sites that are TAZ specific (more) or YAP specific (fewer). One might get the impression that this is an entirely novel concept. As a matter of fact, there are quite a number of earlier studies that showed differences between the transcriptional output of YAP vs TAZ in the same cell context, some including also ChIP-seq data . The authors are advised to relate to that prior knowledge in their Discussion.Reviewer #2:I am not supportive of this manuscript because:1) Above all, the message is hardly novel. There is a number of papers reporting the connections between YAP and TAZ and AP1. With experiments carried out with ChIPseq, comparably to what they did here. The field has also already offered redundant in vitro and in vivo evidence indicating that this cooperation is functional and relevant, including assays transgenic mice with phenotypes associated to YAP hyperactivation. The general conclusion from those papers is that AP1 supports YAP/TAZ function, mainly through TEAD; YAP/AP1 as a subset of YAP/TAZ biology. This is not really the case here. From this perspective, the paper is lagging behind its field.2) A lot of emphasis is placed in 1 cell line, that is an otherwise poorly detailed Scr-inducible model. Strangely, yap/taz KO in these cells is reported to be inconsequential for cell proliferation. Why are they then using siRNA for subsequent assays?3) The role of YAP/TAZ in inducing cell transformation, as defined by growth in soft agar or suspension is already well established. It is an assay for the field, not a result as implied here .4) Transient knockdown of YAP and/or TAZ but not JUNB, strongly decreases STAT3 phosphorylation at Tyr during transformation . Why is it so? this is a description, with no mechanism.5) The biological significance of the mutual requirements shown in Figure 2 on gene expression has no biological validation. Please keep into account that YAP/TAZ knockout mice are readily available to many laboratories and commercially available; so some in vivo validation of some of these claims would not be an absurd request from the journal and an expectation of their readers.6) In Figure 3, the association between YAP and Jun has been already described. The interaction with Stat3 is more interesting, but not developed, and with unclear biological relevance compared to what we already know, from the prior art, on the YAP-Tead and YAP/Jun association.7) the WW Domains of YAP and TAZ are crucial for their interaction with a vast number of proteins. Concluding from their deletion that the WW domains are important for STAT2 and Jun transformation is incorrect.8) the fact that YAP and TAZ differs in their binding to DNA may or not be relevant. There is no biology supporting that they differ in specific functions. This is again merely descriptive.9) Conclusions (from the ER-Src cells) that TEAD is only responsible for 30% of YAP binding sites is at odd with other ChIP seq studies collectively carried out in various cell lines reporting that TEAD is the dominant factor for YAP/TAZ recruitment to DNA. There are likely technical explanations for such discrepancy, such as pull-down efficiency of various transcription factors, or relative expression of TEAD isoforms or differences in the bioinformatic pipelines. Yet the onus is on them to reconcile their work with the rest of the field.10) in figure 6 the manuscript continues to provide descriptions with no biological explorations or even validations that the inferred gene expression relate to at least one of the many biological effects associated to YAP in vitro or in vivo.11) Finding that a gene set for YAP/TAZ activity is able to predict outcome in breast cancer patients is just expected in light of several publications reporting the same conclusion. Showing in Fig7 and Supplement that LumA, B or HER2 show no difference in survival is at odd with a vast number of reports showing that LuminalA tumors have much more favorable outcome than other tumor types , raising questions on their analyses.Reviewer #3:The Authors provide convincing biochemical data supporting the presence of intracellular complexes comprising YAP/TAZ, STAT3 and JUNB in a subset of transformed breast cancer cells.Less clear is what is the genome-wide relationship of these complexes once associated to chromatin and what is the transcriptional relevance of these complexes, given that the genomic analyses, in their present form, do not seem to provide a clear indication of what genes and programs are controlled by these TFs, either alone or in combination.Also, given the all the work is based on the analysis of a limited number of cell lines, the clinical relevance of the YAP/TAZ, STAT3 and JUNB complex is only inferentially supported by the predictive power of a signature of YAP/TAZ, STAT3 and JUNB regulated genes in triple negative breast cancer (but not in other breast cancer subtypes).This may limit the generalization of their proposed model.It is hard to make a point out of the genomic analysis presented, data is analyzed in ways that are not always canonical and which, in my opinion, prevent their interpretations (see below). Also, some conclusions do not seem to be supported by the data presented (i.e. see below the evidence showing that TAZ binding to JUNB and STAT3 is independent from its WW domain). Finally, a strong limitation of this study is the use of only two breast cell lines, one of which is a model for in vitro transformation and not a breast cancer cell line.(1) JUNB is induced by Src, is it also required for Src dependent transformation? Based on the Authors conclusion one might expect that coregulation by YAP/TAZ/JUNB on selected genes is responsible for transformation. If so, what are the genes and programs that are directly regulated by these transcription factors (TFs)?(2) Here are some suggestions on how to improve the analyses of genomic data (ChIP-seq):(a) Please generate ranked heatmaps with normalized ChIP-seq signals for YAP, TEAD, TAZ, STAT3 and JUNB for the following classes of genomic sites: (i) YAP/TAZ common, YAP specific and TAZ-specific (ii) STAT3-bound peaks (iii) JUNB-bond peaks. This should be complemented by box-plot analysis of the relative enrichment signals for all the TFs in the above mentioned genomic regions.(b) Please provide Genome browser snapshots to show quality and enrichment of TF-ChIP-seq signals on relevant genes, as canonical YAP/TAZ targets (CTGF/Cyr612), canonical STAT3 targets and canonical JUNB targets, along with representative genes co-regulated by the four transcription factors.(3) Figure 1 reports growth assays in low attachment or low adhesion. This is meant to demonstrate that loss of YAP, TAZ or both limits growth only when cells are cultured in suspension. Text and figure legend do not report essential details as for instance at what time relative growth was measured. Therefore it is difficult to evaluate the results. Also, it is unclear which cell line was used, the Authors refer to it as "our src-inducible model", I would suggest a more informative name and reference, (for instance MFC10A-SrcER if this was the cell line used). Also, I think the formal interpretation of the results should be that YAP/TAZ are important for anchorage independent growth, rather than transformation. Some of the data presented is questionable: from figure 1E it appears that 2 hrs of tamoxifen are sufficient to lead to a three-fold increase in relative growth, (as compared to ctrl cells treated with EtOH): this seems unrealistic given that in cultures cells typically divide every 14-24 hours.(4) Figure 1 and 2: information concerning replicates of WB experiments is missing. Also, Authors need to evaluate WB signals by densitometric analysis.(5) The CoIP experiments shown in Figure 3A and 3B are of outstanding quality, I would ask to provide further details in the material section .(6) Figure 3D. I disagree with the Authors conclusions: TAZ-DeltaWW1 still binds JUNB and STAT3 with the same efficiency of wt-TAZ as clearly visible in the panel shown here. Also, please provide information of how the coIP was performed .(7) Line 195-203: Authors used peaks summit distance to deduce whether TFs are binding to the same or different DNA sequences, for example they state that since YAP and TAZ ChIP-peaks summits are 25 bp apart, this indicates that YAZ/TAZ are binding the same DNA region. On the other hand, since the distance between STAT3 peaks (or JUNB peaks) and YAP/TAZ peaks is 30-44 bp, they deduce that these TFs are binding close but different DNA sequences. I am not aware of any metrics that allows these deductions, if these are based on prior knowledge, please cite the appropriate reference. Also, all these reasonings seem to be contradicted by later observations (line 235-237) where the Authors conclude that TEAD, AP1 and STAT3 TFs motifs are centered at YAP/TAZ peak summits.(8) Figure 4B. color scale is missing. Also, what are the signals reported in the heatmaps? ChIP-seq signals for YAP and TAZ should be clearly indicated.(9) Figure 4C. The description of this figure is cryptic, it is unclear what TF and KO-confirmed means. As it is, it reads as if out of the 495 TAZ specific peaks, 426 are still detected in TAZ-KO (in EtOH cells). I think the Authors mean that 426 peaks are lost in the TAZ-KO.(10) Figure 5 A. Is this the intersection of ChIP-seq peaks? Please specify in the legend.(11) Figure 6, suppl3: it is surprising the high number of YAP or TAZ peaks, not overlapping TEAD, this is not in line with previous observations. I ask the Authors to comment on this point. Are this really TEAD independent binding or a result of poor performance of the TEAD ChIP-seq?(12) YAP/TAZ/STAT3/JUNB signatures are predictive of poor survival in TNBCs, what are these gene? Are they enriched in any particular pathway or process?eLife. Your article is being evaluated by 1 peer reviewer, and the evaluation is being overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor.Thank you for resubmitting your article entitled "YAP and TAZ are transcriptional co-activators of AP-1 proteins and STAT3 during breast cellular transformation" for peer review at Reviewer #4:SummaryThe YAP and TAZ proteins are the effectors in the HIPPO signaling cascade, serving as co-activators recruited by sequence-specific transcription factors (TFs) such as TEAD proteins. This manuscript shows that YAP and TAZ play a role in transformation of breast epithelial cells, specifically in the context of Src-inducible transformation in an MCF10A cell line. Extensive analyses of protein interactions in solution, genomic TF occupancy profiles (ChIP-seq), and differential expression upon depletion of the TFs provide new insights into the mechanisms of YAP and TAZ recruitment, adding JUNB and STAT3 as recruiters, and the potential roles of the YAP-TAZ-TF complexes recruited by different motifs.Strengths of the manuscript:(1) The experiments establish a role for YAP and TAZ in a specific cell transformation context.(2) The manuscript expands and clarifies the mechanisms of recruitment of the YAP and TAZ co-activators. These co-activators are recruited not only by TEAD TFs, but also by JUNB (a subunit of AP1 family TFs) and STAT3. Previous work had shown recruitment by JUNB (AP1) in the context of a composite binding sites with TEAD TFs, and this manuscript shows recruitment by JUNB without a TEAD binding site motif. Multiple lines of evidence support this conclusion.(3) The manuscript shows that all five (and possibly more) factors  co-occupy many sites, both in the MCF10-inducible Src system and in a breast cancer cell line. Notably, that multi-TF complex was found at genomic intervals with binding site motif instances for single factors, indicating that the complex with recruited co-activators can be directed to distinctly different sets of targets (defined by the binding site motifs present).(4) A series of RNA-seq determinations and analyses on cell lines with the several TFs and co-activators knocked down show that distinct sets of genes are regulated by recruitment of YAP-TAZ to the sites with each specific "recruiting motif".(5) After defining gene sets by the apparent recruitment of the YAP-TAZ complex via AP1 or TEAD motifs, the manuscript shows that high expression of those gene sets are associated with poor prognosis in patients with triple negative breast cancer, using data from TCGA.Weaknesses of the manuscript:(1) While the manuscript expands our knowledge of events, recruitment, and targets in YAP-TAZ co-activation, the biological roles of the different classes of YAP-TAZ complexes and the distinct gene sets that are targets of those different classes are not explored deeply. Some enrichments for gene ontology terms are presented. I realize that this issue was brought up in the initial round of reviews, and the authors argue that further biological analyses are beyond the scope of this study. One wonders what those biological roles may be, and hopefully they will be the subject of further study.(2) Some points in the text and panels in the figures need to be clarified. Details are in the Recommendations for the Authors.Recommendations for the Authors(1) Lines 96-97: The manuscript should explain the rationale for the focus on YAP and TAZ in the epithelial cell transformation system. The Introduction summarizes prior work on an epigenetic switch that drives breast cell transformation via an inflammatory network. However, no explicit statement is provided for why the Hippo pathway (YAP and TAZ) might be involved in this transformation.(2) Lines 97-98: While the Src-inducible system has been used extensively, a brief summary would benefit readers who are not familiar with it. After reading in other sources, I learned that MCF-10A cells are not tumorigenic and presumably not transformed, and I inferred that activation of a (constitutively?) expressed ER-SRC protein will induce transformation. A brief description would be a helpful addition.(3) Lines 118-119: The manuscript should clarify this conclusion and the data that led to it. Perhaps the key observation is that the levels of YAP and TAZ proteins in the three compartments do not change upon activation of the Src transformation protein with tamoxifen. If so, then a sentence stating that observation should be added. Then one can infer that the effects of YAP and TAZ on those AP1 and STAT3 activities just described occur without changes in the YAP or TAZ protein levels in the nucleus.(4) Fig. 3B: Both the upper and lower sets of co-IP assay results are labeled as YAP as the antigen for immuno-precipitation. Is it possible that TAZ was the antigen for the lower set?(5) Fig. 7B indicates some similarity in differential expression upon interference with the five factors, but it is hard to understand and it does not provide quantitative data to support the conclusion of "significant overlap". The legend say that each column corresponds to genes, but the labels on the heatmap indicate that the columns may be the differential expression in each interference condition, with expression for individual genes shown along each row. However, that should produce four discrete values along each row, but instead the color intensity changes along the row. It is not clear why the shades of red and blue change continuously if what is shown is a genes x cell condition matrix.The number of genes in the various possible overlapping sets could be displayed in a clearer manner. For example an "upset" plot can give a readily understood, and quantitative, summary of the overlaps.(6) Line 368 has an odd punctuation; the phrase may be "... site of all ..."(7) Line 392: The p-values are larger than the common threshold of 0.05, and thus it would be more accurate to state something like "The results for both TEAD gene set were not significant" rather than "Both TEAD gene sets have marginal significance". Reviewer #1:The authors have used an MCF10A-based inducible Src transformation model to study the contribution of the YAP and TAZ transcriptional coactivators to cell transformation. YAP and TAZ are transcriptional coactivators and terminal effectors of the Hippo pathway. They don't possess direct DNA binding activity, and are generally believed to be recruited to specific genomic DNA sites through "piggybacking" on TEAD family DNA binding proteins. The authors now report that YAP and TAZ can be recruited to chromatin through direct interactions with AP-1 proteins  and STAT3. They further provide evidence that these interactions are important for cell transformation, and may be conducive to worse patient outcome in triple negative breast cancer (TNBC). They also show that while YAP and TAZ share many binding sites on the genome, each of them has also some "private" sites, associated with distinct classes of genes.Strengths:The findings are interesting and shed new light on transcription regulation by YAP and TAZ. In particular, they provide evidence that – at least in the setting of the studied cell transformation model – the reported interactions may be more impactful than the canonical interaction of YAP/TAZ with TEAD family members. The experiments are well performed and very clearly described.Weaknesses:This study has no major weaknesses. Yet, there is room for improving it. Some suggestions are listed below.1.The data in Figure 6B raise the interesting possibility that CEBP family proteins may serve to recruit YAP/TAZ to a subset of binding sites. This is a novel and potentially valuable finding. Regrettably, the authors do not pursue it further by suitable experimental work. The authors show later that YAP/TAZ are not recruited to CEBP motifs in MDA-MB-231 cells; perhaps this putative interaction is therefore deemed by the authors irrelevant for TNBC and therefore less worthy of pursuing? It should be noted, however, that MDA-MB-231 is a mesenchymal TNBC cell line, and as such it is not representative of the majority of TNBC cases. This reviewer feels that the lack of follow-up on the CEBP lead is a missed opportunity, and such follow-up will make the paper stronger and more innovative. Do YAP/TAZ interact directly with CEBP family members? And is their binding to the indicated sites abrogated by depletion of such CEBP proteins?We agree that the connection of YAP/TAZ to CEBP is potentially interesting. However, additional work on CEBP is well beyond the scope of the present paper and would represent a new and major effort. First, this initial observation has nothing to do with the main conclusions of the paper, and it isn’t mentioned in the abstract. Second, to follow this up properly , it would require a near-equivalent amount of work to what is in the paper for AP-1 and STAT3. Third, an interesting aspect of the CEBP connection is cell-type specificity. Presumably, reviewers would want us to understand the basis of this, which is even more work. Fourth, some reviewers would devalue work on CEBP by saying that it only applies to one cell line and not another.2. The data presented in this study support a model where YAP and TAZ are recruited to AP-1 sites independently of TEAD. However, several earlier studies report that YAP/TAZ transcriptional interaction with AP-1 does occur in concert with TEAD . This should be discussed more thoroughly in the Discussion.As suggested, we have a much more thorough discussion of models. Indeed, we propose a new model  that is clearly different from the Piccolo model.3. In view of published work by the Piccolo group showing the prominence of YAP/TAZ binding to enhancers, the authors may wish to compare the patterns of YAP/TAZ binding in concert with AP-1, STAT3 and CEBP between TSS-proximal (promoter) sites and distal enhancer sites.As suggested, we compare the different types of YAP/TAZ sites in terms of their location. All of them are strongly biased to distal regions, but the different classes of YAP/TAZ sites behave similarly. As shown in the original version, the YAP- and TAZ-specific sites are very strongly biased in the opposite manner, namely much more proximal than distal. This atypical behavior of YAP- and TAZ-specific sites indicates their functional differences from the shared sites.4. The authors may wish to elaborate in more depth and detail on the integration of the chromatin binding site analyses and the functional implications of the transcriptional output of YAP/TAZ/JUNB vs YAP/TAZ/STAT3 vs YAP/TAZ/TEAD.The relationship between protein binding and transcriptional output is shown in Figure 7C. In accord with many studies over the past 15 years, there is a clear connection between binding and transcription, but there are many cases where there is binding, but no transcriptional effect and conversely a transcriptional effect but no binding. In addition, as there are many common genes that are differentially affected by all the factors tested , there isn’t much more to say.5.Please define the workflow of experiments in greater detail. For instance, in Figure 2A, how long before TAM induction of SRC was siRNA knockdown performed? Also, please define "standard conditions of high attachment". Similarly, figure legends should be more technically informative.As stated in the methods, siRNA treatment was for 24 hr prior to adding tamoxifen for the indicated times. “High attachment” conditions are simply normal plates. Comments incorporated into methods and figure legends.6. Figure 3A. According to Figure 2A, STAT3 protein levels do not increase in response to SRC induction. However, "summing up" the protein levels in "E" vs "T", it does look like STAT3 levels increase. Total lysate (input) should be presented. In either case, it would be advantageous to see subcellular localization also by immunofluorescence.Total lysate was presented in Figure 2A. “Summing up” STAT3 protein levels in Figure 3A in the manner described is inappropriate because the amounts of protein loaded in the different lanes are different and are not designed to make a measurement of total STAT3, which was directly done in Figure 2A.7. Was this overexpression and Co-IP experiment performed in MCF10A-ER-SRC cells induced by TAM? If yes, this should be clearly stated.Co-IP experiments in Figure 3A involve endogenous proteins; no overexpression is involved. We clarified this in the figure legend.8. Line 130. "the WW domain of TAZ [is] critical for the interaction with STAT3 and JUNB" Actually, for the Co-IP experiments presented in Figure 3B and 3D, it looks like TAZ interacts only weakly (and certainly more weakly than YAP) with JUNB and STAT3. Is this the case? Is this differential YAP/TAZ behavior represented in the ChIP data? .The co-IP experiments involving YAP vs. TAZ are not suitable for quantitation of interaction strength with JUNB and STAT3 for many reasons, and differences in band intensities are subtle in any event. Similarly, ChIP experiments can’t measure absolute differences between YAP and TAZ binding, but the results are remarkably similar (correlation ~ 0.7), both qualitatively and quantitatively. Indeed, for most analyses, we combine shared YAP and TAZ sites  for simplicity.9. Figure 5D and E. Is there no augmented binding of YAP/TAZ/JUNB or STAT3 on the IL6 enhancer following TAM treatment?In Figure 5D, E , the results for YAP, TAZ, and JUNB are expected from the genome-wide ChIP analysis showing a similar number of sites with similar binding signals. We don’t have a good explanation for why STAT3 binding doesn’t increase upon tamoxifen.10. Figure 6A and line 231. It will be helpful to mention that "JUNB" in this figure is synonymous with AP-1 motif.We clarify that the JUNB and AP-1 motif is the same.11. The difference and meaning of Figure 6—figure supplement 4B and 4C is not explained well enough.We agree that Figure 6-supplement 4B, C was confusing and simply eliminated it, as it had minimal value.12. Figure 7C is not mentioned in the text. Anyway, what does it add?Figure 7C was mentioned in the original text, but it was rather opaque. It has been replaced by new Figure 7B, a histogram of transcriptional profiles upon siRNA knockdowns, to make the point that the various knockdowns give very similar transcriptional profiles. This result is similar to that in our previous paper  that involved multiple DNA-binding transcription factors; YAP/TAZ behave similarly.13. Figure 7E-G. To better assess the impact of YAP/TAZ on STAT3/JUNB function and their relationship to TNBC survival, Kaplan Meier plots should be generated for STAT3/JUNB alone vs STAT3/JUNB/YAP/TAZ.As suggested, we have performed independent Kaplan-Meyer analyses for the various classes of YAP/TAZ sites.14. The authors report that in addition to many target sites shared between YAP and TAZ, there also exist sites that are TAZ specific (more) or YAP specific (fewer). One might get the impression that this is an entirely novel concept. As a matter of fact, there are quite a number of earlier studies that showed differences between the transcriptional output of YAP vs TAZ in the same cell context, some including also ChIP-seq data . The authors are advised to relate to that prior knowledge in their Discussion.Regarding YAP- and TAZ-specific sites, this is a novel conclusion. See point 3 in the novelty/advance section for a more detailed explanation.Reviewer #2:Review 2 is replete with inconsistencies, bias, and negative innuendo ; it should be discarded. Though blunt, this rebuttal will focus solely on the statements in the actual review. It is not a personal attack on Reviewer 2, and I don’t believe that Review 2 is a personal attack on me.I am not supportive of this manuscript because:1) Above all, the message is hardly novel. There is a number of papers reporting the connections between YAP and TAZ and AP1. With experiments carried out with ChIPseq, comparably to what they did here. The field has also already offered redundant in vitro and in vivo evidence indicating that this cooperation is functional and relevant, including assays transgenic mice with phenotypes associated to YAP hyperactivation. The general conclusion from those papers is that AP1 supports YAP/TAZ function, mainly through TEAD; YAP/AP1 as a subset of YAP/TAZ biology. This is not really the case here. From this perspective, the paper is lagging behind its field.This “above all, the message is hardly novel” criticism is incorrect and illogical. We agree that “there are a number of papers reporting connections between YAP/TAZ and AP-1”, and indeed we cited the Piccolo work . However, the YAP/TAZ/AP-1 connection per se is NOT the message of our paper. Instead, the novelty of our work is mechanistic, namely a new molecular model (direct co-activation by AP-1 and STAT3) and different modes of YAP/TAZ recruitment that doesn’t involve TEAD binding to its motifs. Indeed, Review 2 agrees with us by saying that the current view is that “AP-1 supports YAP/TAZ function, mainly through TEAD”, and that “this is not really the case” in our paper. It is illogical to claim that our paper is “hardly novel” yet doesn’t agree with the conventional view. And how can a paper that makes novel conclusions that differ from the conventional view be viewed as “lagging behind the field”? This misrepresentation of “our message” permeates the entire review.2) A lot of emphasis is placed in 1 cell line, that is an otherwise poorly detailed Scr-inducible model. Strangely, yap/taz KO in these cells is reported to be inconsequential for cell proliferation. Why are they then using siRNA for subsequent assays?The statement that we use a “poorly detailed Src-inducible model” is unwarranted as we have published 26 papers using this model, resulting in >5000 citations. Furthermore, this gratuitous comment has nothing to do with the actual results or conclusions of the paper; it is just negative innuendo.On a different point, it is not “strange” that we do siRNA experiments instead of knockouts. Knockouts involve long-term culture, and cells often adapt to the new genetic condition. siRNA (and other depletion) experiments are better for looking at more immediate effects of the genetic perturbation, even though depletions are not complete. The two approaches are not equivalent, and both have value. For example, in our work on yeast Cyc8-Tup1 co-repressor , depletion experiments were critical for elucidating the molecular mechanism that differed from the previous models that were based on knockouts.3) The role of YAP/TAZ in inducing cell transformation, as defined by growth in soft agar or suspension is already well established. It is an assay for the field, not a result as implied here It is well known that YAP/TAZ is important for cell transformation and cancer; that is why we were interested in its role in our model. Figure 1 is simply the obvious control showing that YAP/TAZ is important in our model. To imply that we claim this as novel and an important result of the paper is negative innuendo.4) Transient knockdown of YAP and/or TAZ but not JUNB, strongly decreases STAT3 phosphorylation at Tyr during transformation . Why is it so? this is a description, with no mechanism.The fact that transient knockdown of YAP and/or TAZ on STAT3 phosphorylation  is simply to show a connection between YAP/TAZ and STAT3. This was hardly a major point of the paper, and we never emphasized it. To imply that we claim this as an important result that is critical for any major conclusion of the paper is negative innuendo.5) The biological significance of the mutual requirements shown in Figure 2 on gene expression has no biological validation. Please keep into account that YAP/TAZ knockout mice are readily available to many laboratories and commercially available; so some in vivo validation of some of these claims would not be an absurd request from the journal and an expectation of their readers.As explained above, this paper is concerned with mechanism, not biology, so the issue raised is irrelevant to the central conclusions. It is also unclear what experiment is being suggested (other than a vague statement about using knockout mice). Moreover, it is highly likely that results with knockout mice will vary considerably, depending on which cell types are analyzed and what experimental conditions are chosen. By this logic, the mouse knockout experiments will either validate or invalidate the results in our model depending on which cell types and conditions are used. Also, as discussed before, all the biological experiments performed by others are equally consistent with our model and the Piccolo model.6) In Figure 3, the association between YAP and Jun has been already described. The interaction with Stat3 is more interesting, but not developed, and with unclear biological relevance compared to what we already know, from the prior art, on the YAP-Tead and YAP/Jun associationPerhaps I’m missing something, but I’m unaware of a direct YAP/TAZ interaction with Jun (or other AP-1 family member) or STAT3. This would require the use of purified proteins in addition to co-IP, both of which are presented in our paper. Furthermore, multiple additional lines of evidence indicate that the YAP/TAZ interactions with AP-1 and STAT3 can be independent of TEAD binding to its cognate motif. How else does one explain 1) the YAP/TAZ target sites that lack TEAD motifs, 2) the importance of YAP/TAZ on transcriptional activation on an AP-1 reporter construct (lacks TEAD motifs), and 3) YAP/TAZ binding and co-binding with JunB  to an AP-1 reporter? These results are not explained by the Piccolo model.7) The WW Domains of YAP and TAZ are crucial for their interaction with a vast number of proteins. Concluding from their deletion that the WW domains are important for STAT2 and Jun transformation is incorrect.We are aware that the WW domains interact with multiple proteins and hence that the WW deletion results are only consistent with, not proof, that the interactions with JunB and STAT3 are important for transformation. We have further softened the wording in the manuscript, and never claimed proof. It should be noted that the WW domain deletions still interact with TEAD at normal levels, yet these derivatives are defective in transformation. This indicates that the TEAD interaction is not sufficient for transformation, thereby indicating that other interactions of the WW domain are important. This is a minor aspect of the paper.8) The fact that YAP and TAZ differs in their binding to DNA may or not be relevant. There is no biology supporting that they differ in specific functions. This is again merely descriptive.It is unclear what is meant by “biology”. However, aside from demonstrating YAP- and TAZ-specific direct targets , we provide strong evidence for distinct functions of these specific sites. First, YAP- and TAZ-specific sites are associated with specific, yet different, classes of genes. Second, TAZ-specific sites are enriched for unique motifs suggesting that they are recruited by different transcription factors, which of course have different functions. Third, YAP- and TAZ-specific sites are highly biased to proximal locations, the opposite of YAP/TAZ sites that are highly biased to distal sites. These specific functions occur far beyond random expectation and hence are evolutionarily selected, so it is highly unlikely that they are “irrelevant”.9) Conclusions (from the ER-Src cells) that TEAD is only responsible for 30% of YAP binding sites is at odd with other ChIP seq studies collectively carried out in various cell lines reporting that TEAD is the dominant factor for YAP/TAZ recruitment to DNA. There are likely technical explanations for such discrepancy, such as pull-down efficiency of various transcription factors, or relative expression of TEAD isoforms or differences in the bioinformatic pipelines. Yet the onus is on them to reconcile their work with the rest of the field.The comment that “the onus is on us to reconcile our work with the rest of the field” is anti-scientific. This medieval viewpoint would block new findings and allow published work to go unchallenged. On the contrary, the onus is on Reviewers to review the paper at hand. If they disagree with the conclusions, they must make reasoned arguments based on what is presented. Review 2 doesn’t do this at all.Furthermore, the specific comments about pull-down efficiency, TEAD isoforms, and bioinformatic pipelines do not make sense, because the key observation is based on motifs, not JUNB and TEAD binding. The empirical result that only 30% of YAP/TAZ target sites have TEAD motifs is based on ChIP-seq data with 3 independent antibodies  that give near-identical results. Is Review 2 suggesting that 30% of our data is correct and 70% is incorrect? Or is Review 2 suggesting that we don’t know how to perform and analyze ChIP-seq experiments and do motif analysis?Lastly, the comment is incorrect because the paper explicitly says that we find examples that fit the Piccolo model of composite sites. However, the results clearly show that most YAP/TAZ sites do not have TEAD motifs and hence cannot be explained by the Piccolo model. More generally, comment 9 is strongly suggestive of bias, which  is why I request that this review be discarded.10) in figure 6 the manuscript continues to provide descriptions with no biological explorations or even validations that the inferred gene expression relate to at least one of the many biological effects associated to YAP in vitro or in vivo.I don’t understand what “no biological explorations or even validations that the inferred gene expression relate to at least one of the many biological effects” is supposed to mean. Figure 6 is concerned with motif analysis of YAP/TAZ target sites, a mechanistic issue totally unrelated to this comment.11) Finding that a gene set for YAP/TAZ activity is able to predict outcome in breast cancer patients is just expected in light of several publications reporting the same conclusion. Showing in Fig7 and Supplement that LumA, B or HER2 show no difference in survival is at odd with a vast number of reports showing that LuminalA tumors have much more favorable outcome than other tumor types , raising questions on their analyses.Review 2 misses the point about the survival analysis . This novelty of this analysis is that it specifically examines different classes of YAP/TAZ target genes, something that could not be done previously because such target genes are identified in our paper. This analysis provides biological importance to the molecular mechanism (direct co-activation via AP-1 and STAT3) that is the central conclusion of the paper. Thus, our results are new and hence not “just expected” or “at odds with a vast number of reports” (how could they be both?). If Review 2 wishes to question our standard analysis, it is necessary to address the analysis itself. Lastly, the “established clinical fact” that luminal A tumors have more favorable outcome than other tumor types is unrelated to our analysis and hence irrelevant. We are not comparing tumor types for patient outcomes, but rather whether a set of molecularly defined genes is linked to patient outcomes.Reviewer #3:The Authors provide convincing biochemical data supporting the presence of intracellular complexes comprising YAP/TAZ, STAT3 and JUNB in a subset of transformed breast cancer cells.Less clear is what is the genome-wide relationship of these complexes once associated to chromatin and what is the transcriptional relevance of these complexes, given that the genomic analyses, in their present form, do not seem to provide a clear indication of what genes and programs are controlled by these TFs, either alone or in combination.Also, given the all the work is based on the analysis of a limited number of cell lines, the clinical relevance of the YAP/TAZ, STAT3 and JUNB complex is only inferentially supported by the predictive power of a signature of YAP/TAZ, STAT3 and JUNB regulated genes in triple negative breast cancer (but not in other breast cancer subtypes).This may limit the generalization of their proposed model.General Comments: It is hard to make a point out of the genomic analysis presented, data is analyzed in ways that are not always canonical and which, in my opinion, prevent their interpretations (see below). Also, some conclusions do not seem to be supported by the data presented (i.e. see below the evidence showing that TAZ binding to JUNB and STAT3 is independent from its WW domain). Finally, a strong limitation of this study is the use of only two breast cell lines, one of which is a model for in vitro transformation and not a breast cancer cell line.See comment 4 in the novelty/advance section about the predominant use of a single cell line.(1) JUNB is induced by Src, is it also required for Src dependent transformation? Based on the Authors conclusion one might expect that coregulation by YAP/TAZ/JUNB on selected genes is responsible for transformation. If so, what are the genes and programs that are directly regulated by these transcription factors (TFs)?per se, but various classes of YAP/TAZ target genes are discussed in the paper.In our model, JUNB is one of many examples where the gene is induced by Src (and Ras and other stimuli) and important for transformation. This has been extensively studied in many of our previous papers, and indeed is the basis of the epigenetic switch that relies on an inflammatory positive feedback loop. The present paper doesn’t add much on the inflammatory loop (2) Here are some suggestions on how to improve the analyses of genomic data (ChIP-seq):(a) Please generate ranked heatmaps with normalized ChIP-seq signals for YAP, TEAD, TAZ, STAT3 and JUNB for the following classes of genomic sites: (i) YAP/TAZ common, YAP specific and TAZ-specific (ii) STAT3-bound peaks (iii) JUNB-bond peaks. This should be complemented by box-plot analysis of the relative enrichment signals for all the TFs in the above mentioned genomic regions.(b) Please provide Genome browser snapshots to show quality and enrichment of TF-ChIP-seq signals on relevant genes, as canonical YAP/TAZ targets (CTGF/Cyr612), canonical STAT3 targets and canonical JUNB targets, along with representative genes co-regulated by the four transcription factors.Best reviewer comment I’ve received in years. We did the suggestion as well as follow-up analyses, and obviously, the manuscript has changed for the better, including a different (and novel) molecular view of what is going on. We don’t think a Gene Browser snapshot for a single locus adds much given the new Figure 5A, which is far more relevant and informative.(3) Figure 1 reports growth assays in low attachment or low adhesion. This is meant to demonstrate that loss of YAP, TAZ or both limits growth only when cells are cultured in suspension. Text and figure legend do not report essential details as for instance at what time relative growth was measured. Therefore it is difficult to evaluate the results. Also, it is unclear which cell line was used, the Authors refer to it as "our src-inducible model", I would suggest a more informative name and reference, (for instance MFC10A-SrcER if this was the cell line used). Also, I think the formal interpretation of the results should be that YAP/TAZ are important for anchorage independent growth, rather than transformation. Some of the data presented is questionable: from figure 1E it appears that 2 hrs of tamoxifen are sufficient to lead to a three-fold increase in relative growth, (as compared to ctrl cells treated with EtOH): this seems unrealistic given that in cultures cells typically divide every 14-24 hours.We have described and used the low-attachment assay is multiple papers, but as suggested, we add more methodological details. Growth in soft agar has been the standard transformation assays for 6 decades, and our low attachment assay is just a different version of the same assay (both are presented). In addition, we clarify the 2-hr experiment. After the 2-hr tamoxifen treatment, the cells are seeded on low attachment plates (thereby removing the tamoxifen) and cells grow for 4 days before being assayed. The Reviewer may not be aware of our initial paper on this system  showing that short-term tamoxifen treatment is sufficient for transformation .(4) Figure 1 and 2: information concerning replicates of WB experiments is missing. Also, Authors need to evaluate WB signals by densitometric analysis.As suggested, we have quantitated WB signals.(5) The CoIP experiments shown in Figure 3A and 3B are of outstanding quality, I would ask to provide further details in the material section .We appreciate the comment about outstanding quality. Requested details  provided.(6) Figure 3D. I disagree with the Authors conclusions: TAZ-DeltaWW1 still binds JUNB and STAT3 with the same efficiency of wt-TAZ as clearly visible in the panel shown here. Also, please provide information of how the coIP was performed The reviewer is partially correct. Unlike what we implied in the original paper, the TAZ-WW derivative does interact with JUNB and STAT3, but quantitation (now provided) indicates less well than wild-type TAZ. We have modified the text. All YAP and TAZ derivatives were cloned into pCDNA3.1 plasmid and overexpressed in 293T cells for two days before harvesting for co-IP experiments using anti-HA antibody or IgG control. This is now included in the methods.(7) Line 195-203: Authors used peaks summit distance to deduce whether TFs are binding to the same or different DNA sequences, for example they state that since YAP and TAZ ChIP-peaks summits are 25 bp apart, this indicates that YAZ/TAZ are binding the same DNA region. On the other hand, since the distance between STAT3 peaks (or JUNB peaks) and YAP/TAZ peaks is 30-44 bp, they deduce that these TFs are binding close but different DNA sequences. I am not aware of any metrics that allows these deductions, if these are based on prior knowledge, please cite the appropriate reference. Also, all these reasonings seem to be contradicted by later observations (line 235-237) where the Authors conclude that TEAD, AP1 and STAT3 TFs motifs are centered at YAP/TAZ peak summits.We apologize for confusing language about the pairwise analysis of peak summits. We meant to say that the binding locations of all factors are extremely close together and likely coincide at the relevant motif. We did not unambiguously claim identical binding locations because the distances aren’t exactly the same and there is some error. Also, there may be some subtle effects on exactly where crosslinking occurs. For example, in previous work, we showed the NFYA and NFYB subunits of the heteromeric NFY factor have peak summits 15 bp apart and asymmetric with respect to the CCATT motif .(8) Figure 4B. color scale is missing. Also, what are the signals reported in the heatmaps? ChIP-seq signals for YAP and TAZ should be clearly indicated.(9) Figure 4C. The description of this figure is cryptic, it is unclear what TF and KO-confirmed means. As it is, it reads as if out of the 495 TAZ specific peaks, 426 are still detected in TAZ-KO (in EtOH cells). I think the Authors mean that 426 peaks are lost in the TAZ-KO.(10) Figure 5 A. Is this the intersection of ChIP-seq peaks? Please specify in the legend.Clarified(11) Figure 6, suppl3: it is surprising the high number of YAP or TAZ peaks, not overlapping TEAD, this is not in line with previous observations. I ask the Authors to comment on this point. Are this really TEAD independent binding or a result of poor performance of the TEAD ChIP-seq?The original Figure 6, supplement 3 referred to motifs, not binding. This whole issue is related to point 2 and the major change in the paper.(12) YAP/TAZ/STAT3/JUNB signatures are predictive of poor survival in TNBCs, what are these gene? Are they enriched in any particular pathway or process?The various genes used in the survival analyses are presented in Table S3.