A degradable form of polyoma small T antigen reveals the high specificity of TAZ in regulating gene expression

Significance

Studying tumor-causing viruses and their encoded proteins has shaped modern cancer biology. One viral protein—the polyoma small T antigen (PyST)—has small size and robust interaction with protein phosphatase complexes, making it suitable for tool development. We engineered a degradable version of PyST that maintains its transforming activity. By manipulating the protein abundance of PyST and consequently its effector, the transcription cofactor TAZ, we found that TAZ regulates a surprisingly small number of genes. These limited number of gene targets of TAZ mediate the transforming potential of PyST. Our study thus develops a tool that offers potent regulation of TAZ by a viral oncogene and insights into its roles in controlling gene expression.

Abstract

The study of DNA tumor viruses has revolutionized cancer biology, partly by virtue of the unique capabilities of viral oncoproteins to manipulate key proteins and pathways involved in tumorigenesis. We find a high affinity and selective binding of the polyoma small T antigen (PyST) with the transcription cofactor TAZ. By engineering a degradable version of PyST, we demonstrate that, when TAZ activity is modulated by PyST, a surprisingly small number of genes have altered expression and thus are candidate transcription targets of TAZ. Notably, knocking out TAZ, or its target genes CTGF or CYR61, abolishes the growth-promoting properties of PyST that are evident upon growth factor withdrawal. Therefore, by controlling the protein abundance of PyST and consequently TAZ activity, we find that TAZ is a transcriptional coactivator that can achieve important biological effects by acting on a limited number of gene targets.

YES-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding motif (TAZ), are key transcription control modules of the Hippo pathway (1, 2). They were first discovered via their association with tyrosine kinase YES (3, 4), and the signal modulator protein14-3-3 (5). YAP and TAZ were subsequently found to be involved in transcriptional activation, mediated by the interaction between the WW domains of YAP/TAZ and the Pro-Pro-Xxx-Tyr (PPXY) motifs found in the activation domain of many transcription factors (5, 6).

The predominant transcription factors that mediate the functions of YAP/TAZ are TEAD, proteins with a TEA sequence motif that is shared by several transcription (co-)factors (7). The YAP–TEAD connection was identified by profiling TEAD-interacting proteins in mammalian cells (8) and by screening transcription factors that mediate YAP-dependent induction of gene expression (9). Recent studies sequencing genomic loci bound by YAP/TAZ and/or TEADs have implicated YAP/TAZ in regulating the expression of a few thousand genes (10, 11). While accumulating evidence indicates that YAP and TAZ can target common targets, each one also has unique activities (12). It remains a challenge to develop strategies to modulate YAP/TAZ activity with sufficient selectivity to stratify the YAP/TAZ-regulated genes for their biological impact.

Studying tumor virus antigens helped to pioneer the modern era of biology, leading to a series of discoveries including those of the first oncogenes, tumor suppressor genes, tyrosine kinase, and phosphoinositide 3-kinase (PI3K) activities (13, 14). In recent decades, together with others we have found that murine polyoma small T antigen (PyST) is able to bind to a number of proteins including protein phosphatase PP2A (15, 16), TAZ (17), and YAP (18). In this study, we aimed to achieve a quantitative view of the PyST interactome and to develop a switchable PyST-based tool for modulating TAZ protein abundance. Distinctly different from the traditional view of YAP/TAZ regulating a few thousand genes, our study suggests that the number of genes most sensitively modulated by TAZ could be less than three dozen and that these genes are important for the biological effects of TAZ. We propose that the transcriptional coactivator TAZ can regulate gene expression in a highly specific manner.

Results

PyST Binds to TAZ with a Comparable Affinity to Its Interaction with PP2A.

We and others have previously demonstrated that PyST is capable of binding to the transcription cofactors YAP/TAZ (17, 18). To further understand the interaction, we expressed Flag-tagged PyST (Flag on the carboxyl terminus of PyST) in HEK293T cells and then subjected cell extracts to immunoprecipitation using anti-Flag antibody. In the immunoprecipitates from samples with the expression of wild-type PyST, we observed an apparent enrichment of PyST signal along with TAZ and PP2A (Fig. 1A). Expression of a mutant PyST (R103A)—a mutant we previously found to interrupt PyST–YAP binding (18)—abrogated the interaction between PyST and TAZ while leaving PP2A binding intact (Fig. 1A). To address how the PyST–PP2A–TAZ interaction may impact the phosphorylation of TAZ, we probed the samples with an antibody recognizing phosphorylated TAZ and subsequently with anti-total TAZ. Compared with the input, the immunoprecipitates of PyST had nearly invisible signal of phosphorylated TAZ but showed a strong signal of total TAZ (Fig. 1B). These results suggest that TAZ becomes hypophosphorylated by association with PyST, likely due to PP2A-catalyzed dephosphorylation leading to the stabilization of TAZ protein (as cartooned in Fig. 1C).

Fig. 1.

Polyoma virus small antigen (PyST) binds to PP2A and TAZ and induces TAZ dephosphorylation. (A) HEK293T cells were transfected with pCDH vector encoding GFP, Flag-tagged wild-type or mutant (R103A) PyST. Samples were subjected to immunoprecipitation with anti-Flag (raised in mouse or rat) affinity gel. The resulting immunoprecipitates were subjected to fluorescence immunoblotting using the indicated antibodies (all raised in rabbit except anti-β-tubulin, to avoid signals from existing IgG in the immunoprecipitates). (B) Samples are prepared as in (A) and subjected to immunoblotting using anti-phospho-TAZ (S89) (Top). The same membrane was subsequently probed with anti-total TAZ (Middle). * denotes a nonspecific signal likely derived from the heavy chain of anti-Flag immunoglobin. The white arrows point to phospho- or total TAZ signals in the immunoprecipitates. (C) Cartoon depicting the dephosphorylation and protein stabilization of TAZ by PyST-mediated interaction with and recruitment of PP2A. A, B, and C denote the scaffolding, regulatory, and catalytic subunits of PP2A protein complex. The streaks in the phosphorylated form of TAZ at the top right indicate its instability. (D) Workflow for quantification of PyST-interacting proteins. Note that the four different colors in the cartoon depict four individual samples used in the proteomic analysis, two control and two PyST immunoprecipitation samples. (E) Plot showing the quantification of recovered peptides from the anti-Flag immunoprecipitates. Fold of enrichment is expressed as the signal of the indicated proteins in immunoprecipitates of PyST-expressing samples relative to that in control cells expressing GFP.

We next aimed to characterize PyST–TAZ binding in a quantitative manner. To this end, we immunoprecipitated and then eluted PyST complex and subjected the samples to multiplexed proteomics (Fig. 1D and SI Appendix, Fig. S1A). As expected, we observed a significant enrichment of all subunits comprising the common heterodimeric core enzyme of PP2A, catalytic subunits C (PPP2CA, PPP2CB), and regulatory subunits A (PPP2R1A, PPP2R1B); surprisingly, TAZ demonstrates a similar degree of enrichment as PP2A (Fig. 1E and SI Appendix, Fig. S1B and Dataset S1). The mutant form of PyST (PyST^R103A) maintains the interaction with PP2A, as well as a few other proteins including Lipin-1/2 (19), but has completely lost the interaction with TAZ as revealed by the quantitative mass spectrometric assays (SI Appendix, Fig. S1 C and D and Dataset S2). We also found that the interaction between PyST and TAZ does not rely on the phosphatase activity of PP2A, since briefly exposing cells to a PP2A inhibitor did not abolish the recovery of TAZ protein in the PyST immunoprecipitates (SI Appendix, Fig. S1E). In addition, a PyST mutant (C142S) that is known to have impaired binding to PP2A (18) retained its capability to interact with TAZ (SI Appendix, Fig. S1F). Together, these data indicate a significant interaction between PyST and TAZ, suggesting a model in which TAZ could be recruited by PyST into the proximity of PP2A for dephosphorylation.

Previous studies have shown that SV40 small T antigen promotes the interaction between PP2A A and C subunits with an atypical PP2A complex STRIPAK (20) to regulate HIPPO kinases MST1/2 and MAP4Ks and consequently the status of YAP phosphorylation (21–23). To understand whether PyST may also utilize a similar mechanism to impact TAZ, we compared PyST with SV40 as well as Merkel cell polyomavirus (MCV) small T. All three small T antigens had potent interaction with PP2A A and C subunits; however, unlike PyST, SV40 and Merkel cell polyomavirus (MCV) small T did not bind to TAZ (SI Appendix, Fig. S2A). SV40 small T, not PyST, was found to have interaction with STRIP1, a core component of the STRIPAK complex (SI Appendix, Fig. S2B). Notably, stable expression of PyST in a nontransformed human mammary epithelial cell line MCF-10A cells led to an increased TAZ protein abundance without a decrease in the phosphorylation of Hippo pathway regulators LATS1 or MOB1 (SI Appendix, Fig. S2 C and D). These observations suggest that it is unlikely PyST utilizes STRIPAK to regulate TAZ.

PyST Expression Enhances TAZ Protein Abundance.

We then further characterized PyST-induced TAZ signal. Consistent with the observations that PyST, but not SV40 or MCV ST, readily immunoprecipitated TAZ (SI Appendix, Fig. S2A), the increase of TAZ protein abundance—as detected by two independent monoclonal antibodies—was apparent in cells expressing wild-type PyST, but not robust in cells expressing SV40 or MCV small T (Fig. 2A and SI Appendix, Fig. S3A). Interestingly, neither total nor phosphorylated YAP was apparently altered by this level of polyoma, SV40, or MCV small T expression (Fig. 2A and SI Appendix, Fig. S3A). We also stably expressed PyST in multiple cell lines including HEK293T, MCF-10A, MDA-MB-231, and MDA-MB-468. In all these cells, wild-type PyST expression drove up the protein abundance of TAZ (SI Appendix, Fig. S3 B and E).

Fig. 2.

PyST expression activates TAZ and confers TAZ-dependent cell survival under long-term starvation of growth factors. (A) HEK293T cells were transfected with pCDH-puro vector encoding GFP control or indicated T antigens and, on the next day, subjected to overnight serum starvation, followed by harvest with 1x SDS sample buffer. Total cell lysates were subjected to fluorescent immunoblotting using the indicated antibodies (clone number indicated for monoclonal antibodies). (B) MCF-10A control cells (GFP-expressing), cells expressing PyST, or cells transduced with lentiCRISPR-sgPTEN were seeded and subjected to overnight starvation of serum, EGF, and insulin. Cell lysates were then prepared and used for immunoblotting. (C) MCF-10A cells stably transduced with pCDH-puro lentiviral empty vector control or lentivirus encoding PyST were starved overnight of serum, EGF, and insulin, before being treated with cycloheximide (50 µg/mL) for the indicated time points. Signals of TAZ protein (Top) or MYC (Bottom) were measured by immunoblotting and normalized to those of β-actin. (D) The indicated MCF-10A cells were seeded in 96-well plates (1,000 cells per well) in MCF-10A complete medium (supplemented with serum, EGF, and insulin) or MCF-10A assay medium (supplemented with insulin and half-reduced serum). In 5 (Left) and 10 d (Right), cells were subjected to CellTiter-Glow assays. Raw data of luminescence were plotted. Note that cells have not reached the maximum confluence after 5 d of culture under the mentioned seeding and culture conditions. (E) The indicated MCF-10A cells were assayed as in (B) for fluorescent immunoblotting. Note that cells expressing the PyST^R103A mutant have significantly less abundance of TAZ or CYR61 proteins, compared to cells expressing wild-type PyST. (F) The indicated MCF-10A cells were seeded and, upon reaching visual confluence, were either fixed for crystal violet staining (Top) or subjected to long-term starvation culture. 4 wk or longer after the initial serum/growth factor (GF) starvation, cells were fixed and stained (Bottom). Note that all control cells perished following the starvation, while PyST-expressing cells survived and maintained the confluent monolayer. (G) The indicated MCF-10A cells were starved of serum and growth factors overnight, and lysates were prepared and subjected to fluorescent immunoblotting (Left). The cells were also assayed as in (F) for crystal violet staining (Right Top) and quantification (Right Bottom).

We next compared PyST expression with other known or potential regulators of YAP/TAZ to estimate the potency of PyST in activating TAZ. GPCR activation, such as by serum-borne GPCR ligands, is known to robustly activate YAP and TAZ (24). We first found that the effect of PyST expression on TAZ does not rely on growth factors or GPCR ligands present in serum, because PyST-induced signal was observed under both the normal tissue culture conditions of MCF-10A cells—i.e., supplementation of serum, EGF, and insulin—and upon exposure to medium without any supplemented growth factors (SI Appendix, Fig. S3E). Notably, the effect of PyST expression on TAZ appeared to be comparable to serum-induced activation of the YAP/TAZ pathway (SI Appendix, Fig. S3F). We also compared PyST expression with the activation of phosphoinositide 3-kinase (PI3K), a known mediator of mitogenic growth factors in regulating YAP (25). To this end, we used CRISPR/Cas9-mediated gene editing to knock down the expression of tumor suppressor PTEN to activate the PI3K pathway. We found that PyST expression is significantly more potent than PTEN loss at inducing TAZ protein abundance, as well as CYR61 (CCN1) (Fig. 2B), an established transcriptional target of TAZ (26, 27). In line with the finding that dephosphorylation of TAZ stabilizes the protein (28), we found that in cells stably expressing PyST, the stability of TAZ protein was significantly increased while the short-lived protein MYC was not affected (Fig. 2C and SI Appendix, Fig. S3G). In addition, the stability of TAZ transcript appeared not to be altered by PyST expression (SI Appendix, Fig. S3H). Together these data support the model wherein PyST recruits TAZ and PP2A leading to TAZ dephosphorylation and protein stabilization (Fig. 1C).

PyST Promotes TAZ-Dependent Cell Growth and Survival.

Given the documented, pro-oncogenic roles of YAP/TAZ, we asked whether PyST expression confers cells an advantage in proliferation and/or survival. To this end, we chose MCF-10A cells, a nontransformed human mammary epithelial cell line that has been extensively utilized in studying YAP/TAZ biology. Under standard culture conditions, we did not observe an apparent effect of PyST expression on cell growth (Fig. 2 D, Left). However, when the cells were cultured in the absence of EGF—a known activator of YAP/TAZ (29), cells expressing wild-type PyST exhibited an approximately two-fold increase in cell growth, compared to cells expressing either GFP control or the PyST R103A mutant (Fig. 2 D, Right and SI Appendix, Fig. S4A). Such a pattern of cell growth is consistent with the induction of TAZ and CYR61 expression in cells expressing wild-type PyST (Fig. 2E) and is consistent with previous reports of a relatively weak transforming activity of PyST in NIH 3T3 cells (30–33). We then tested various cell growth conditions and developed a culture model wherein the effect of PyST became profound; under prolonged culture—4 wk or longer—in the absence of any growth factor or serum supplementation, PyST-expressing cells robustly survived while control cells all perished (Fig. 2F and SI Appendix, Fig. S4B). Thus, in the absence of any exogenous growth factors, PyST expression is capable of promoting cell growth and survival, a typical characteristic of many oncogenic events found in human cancers.

To determine the role of TAZ in PyST-induced cell survival, we utilized CRISPR/Cas9-mediated gene editing to knockout TAZ in PyST-expressing cells (Fig. 2G and SI Appendix, Fig. S6A). Compared with wild-type cells expressing PyST (with elevated TAZ protein), PyST-expressing, TAZ-knockout (KO) cells showed significantly reduced growth and survival in the absence of exogenous growth factors (Fig. 2G). Notably, these cells had no apparent growth defects under standard culture conditions (SI Appendix, Fig. S4C). We further reexpressed TAZ in PyST-expressing and TAZ knockout cells and found that these cells restored the expression of TAZ and importantly, their capability to survive under long-term growth factor deprivation (SI Appendix, Fig. S4 D and E). These observations suggest that TAZ, although likely not the sole downstream PyST effector, acts as a key factor in mediating PyST-induced cell growth and survival.

TAZ Abundance Regulated Through Inducible Protein Degradation of PyST.

Considering our finding that TAZ is modulated by PyST with high selectivity and that TAZ plays a major role in the capacity of PyST to promote cell growth and survival, we next attempted to develop a strategy to achieve temporal control of TAZ activity by regulating PyST protein stability. To this end, we fused PyST to FKBP12^F36V, a mutant FKBP12 that has an exceptional affinity for AP1867—a synthetic derivative of the natural product FK506 (34). The resulting PyST/ FKBP12^F36V fusion protein can be regulated by a heterobifunctional degrader consisting of AP1806 and thalidomide (35, 36), for rapidly induced protein degradation (Fig. 3A).

Fig. 3.

Developing a PyST–FKBP12^F36V fusion protein for temporal control of TAZ. (A) Scheme for inducible degradation of FKBP12^F36V-tagged PyST. Sequence of the linker region between PyST and FKPB12^F36V is shown. Various inhibitors used for interfering with CUL4-RING E3 ligase-mediated protein degradation are also noted. (B) HEK293T cells were transfected with the indicated DNA and were harvested for fluorescent immunoblotting after overnight serum starvation. Tagging FKBP12^F36V to the amino-terminus of PyST generated an additional protein of unexplained size and failed to enhance TAZ protein abundance. (C) Parental MDA-MB-468 cells or cells stably transduced with PyST–FKBP12^F36V were pretreated with vehicle control or the indicated inhibitors for 1 h, followed by 2 h of exposure to dTAG13 (200 nM). Cell lysates were then harvested and used for immunoblotting. (D) MCF-10A cells stably expressing the indicated protein were treated with vehicle control or dTAG13 (200 nM) overnight before being harvested for lysate preparation and fluorescent immunoblotting. The merged blots indicate a typical blotting experiment in which the membrane was incubated with two primary antibodies raised in different species (pseudocolor red was used for TAZ, and green for β-actin signal). (E) The indicated MCF-10A cells, upon reaching visual confluence, were either fixed for crystal violet staining (Top Left), or subjected to prolonged starvation of serum and growth factors before cells were stained (Top Right). The graph (Bottom) depicts the quantification of crystal violet staining with the raw absorbance values. (F) Cells were assayed as in (E) except that vehicle control or dTAG13 was added over the course of starvation. Note that dTAG13 treatment abolished the growth and survival advantages conferred by the expression of PyST–FKBP12^F36V.

We fused FKBP12^F36V to either the amino- or carboxyl-terminus of PyST, to determine which tagging position maintains the activity of PyST in regulating the protein abundance of TAZ. In HEK293T cells, we found that PyST–FKBP12^F36V, but not FKBP12^F36V–PyST, increased TAZ protein levels to a similar extent as wild-type PyST (Fig. 3B and SI Appendix, Fig. S5 A and B). This C-terminal fusion protein was also highly sensitive to the heterobifunctional degrader dTAG13 (Fig. 3C and SI Appendix, Fig. S5 C and D). Furthermore, dTAG13-induced protein degradation was completely rescued by cotreatment with proteasome inhibitors MG132 or Calfizomib, lenalidomide to competitively inhibit the degrader, or MLN4924—an inhibitor of the NEDD8-activating enzyme (NAE) (37) that blocks NEDD8 conjugation and the resulting activity of cullin-RING subtypes of ubiquitin ligases (Fig. 3C and SI Appendix, Fig. S5E). In MCF-10A cells stably transduced with PyST–FKBP12^F36V, we observed that dTAG13 efficiently diminished the abundance of the PyST fusion protein and abrogated the induction of TAZ (Fig. 3D). Similar to PyST, PyST–FKBP12^F36V expression was potent in conferring cell growth and survival under long-term growth factor starvation (Fig. 3E), an effect that was sensitive to the treatment of dTAG13 (Fig. 3F).

Altering TAZ Abundance Via Inducible PyST Protein Degradation Impacts the Expression of a Small Number of Genes.

With a nuanced inducible degradation tool for PyST–FKBP12^F36V and, as a result, inducible “inactivation” of TAZ, we proceeded to study the magnitude and scope of gene expression regulated by TAZ in PyST-expressing cells. Due to other possible non-TAZ effects downstream of PyST expression, we considered that any potential effect of gene expression by PyST–FKBP12^F36V might overestimate the effect of TAZ on gene expression control. However, and opposite of the prediction, we found that only a very small number of genes demonstrated significantly altered expression in cells expressing PyST–FKBP12^F36V compared with control MCF-10A cells. Notably, genes with the highest increases in expression included CYR61 (CCN1), CTGF (CCN2), a few early immediate genes (e.g., MYC, EGR1, FOS, JUN), and genes encoding the dual-specific phosphatases (DUSPs) (Fig. 4A). To validate further the specific phenotype observed in gene expression driven by PyST-induced TAZ, we subjected cells expressing PyST–FKBP12^F36V to dTAG13 treatment to degrade the fusion protein and consequently deinduce TAZ protein. In an extremely dramatic manner, those genes whose expression increased in cells expressing PyST–FKBP12^F36V (compared with control cells) now all showed decreased expression upon dTAG13 treatment (Fig. 4B).

Fig. 4.

Inducible regulation of TAZ reveals a surprisingly small set of genes impacted when TAZ protein abundance is deinduced. (A) Volcano plot of gene expression in MCF-10A cells expressing PyST–FKBP12^F36V compared with cells expressing vector control. Cells were seeded, and on the next day, refreshed with medium lacking any serum or growth factors. Following overnight incubation, cells were subjected to total RNA extraction and deep sequencing. Genes with absolute changes in expression greater than 1 (log₂ fold change, FC), adj P-value < 0.1 are colored blue (downregulated) or red (upregulated). The dot plot on the Right illustrates all significantly upregulated genes in cells expressing PyST–FKBP12^F36V compared to control cells. Note that TAZ transcript level is not significantly changed (log₂FC is -0.23). (B) Volcano plot of gene expression in cells expressing PyST–FKBP12^F36V that were exposed to dTAG13 compared to vehicle control. Cells were treated with vehicle (DMSO, 0.02%, v/v) or dTAG13 (200 nM) in medium that was free of serum or any supplemented growth factors. Following 22 h of treatment, cells were subjected to RNA extraction and deep sequencing. All genes significantly downregulated by dTAG13 treatment are illustrated in the graph on the Right. Note that TAZ transcript level is not significantly changed by dTAG13 treatment (log₂FC is 0.14). (C) Effects of acute inhibition of TEAD on gene expression. (Top Left) A schematic illustrating the lipid-binding pocket of TEAD proteins. The sulfhydryl (SH) group side chain of the cysteine, known to be S-palmitoylated, is noted. (Middle Left) Chemical structure of the TEAD inhibitor MYF-03-69, which forms a covalent bond with the aforementioned cysteine in TEAD. The three volcano plots illustrate gene expression changes in NCI-H226 cells treated with MYF-03-69 (6 h, doses as indicated), compared with cells treated with vehicle control. (D) The indicated cells were seeded, and upon reaching visual confluence, were either fixed and stained with crystal violet (Top) or subjected to long-term starvation of serum and growth factors (Bottom). The bar graphs show the quantification of the staining. (E) (Top) Fluorescent immunoblots for lysates from the indicated cells. (Bottom) Cells were subjected to long-term starvation assay as in (D). (F) Crystal violet staining of the indicated cells (Top) and quantification of the staining (Bottom). MCF-10A cells were stably transduced with viral vector control or vectors encoding either CTGF or CYR61. Cells were then subjected to starvation assay as in (D). Indicated P values were derived from Student’s t test.

These results were very unexpected. Previous studies based on chromatin immunoprecipitation followed by sequencing have revealed a few thousand genomic loci bound by YAP/TAZ, indicating the regulation of potentially a large number of genes by YAP/TAZ (10, 11, 38, 39). Thus, it was quite unexpected that cells expressing PyST, and, therefore, increased protein abundance of TAZ, could only have less than three dozen genes whose expression is different from that in control cells. Interestingly, in mesothelioma cells that depend on YAP/TAZ/TEAD pathway for proliferation, treatment of a covalent TEAD inhibitor also resulted in alterations in a small number of genes (Fig. 4C; 40), lending support to the observations based on the use of degradable PyST as a tool to modulate TAZ. Indeed, when we treated PyST-expressing MCF-10A cells with the TEAD inhibitor, only a very small number of genes demonstrated reduced expression (SI Appendix, Fig. S7 A and B).

Since there are only a small number of genes whose expression is sensitive to PyST-induced TAZ, we then asked whether those genes may mediate the ability of PyST to promote cell growth and survival in the absence of growth factors. We used CRISPR/Cas9 gene editing approach to knock out CTGF and CYR61 in cells expressing PyST (SI Appendix, Fig. S6 B and C). Interestingly, PyST-expressing cells with either CTGF or CYR61 knocked out behaved like control cells, and all perished during prolonged growth factor starvation (Fig. 4 D and E). We next overexpressed TAZ in MCF-10A cells and found that TAZ overexpression was insufficient to confer cell growth and survival advantages (SI Appendix, Fig. S7C); these observations are consistent with its weak effect in inducing the expression of TAZ target genes such as CYR61 (SI Appendix, Fig. S7D) and also suggest that PyST could have additional activities coming directly from its effects on TAZ phosphorylation or indirectly from its other activities. We also overexpressed CYR61 and CTGF in MCF-10A cells to understand whether their expression could be sufficient to phenocopy PyST expression. The overexpression resulted in a limited but statistically significant effect on cell growth and survival (Fig. 4F). Notably, we have not ruled out the possibility that other genes modulated by PyST, such as MYC, may also contribute to this phenotype. Nevertheless, it is notable that only a small number of genes are regulated by PyST-induced TAZ and significantly impact cell growth in the absence of growth factors. Therefore, these observations suggest that the effects of TAZ on gene expression can be highly specific, at least in the context of PyST expression.

Discussion

In this study, we first demonstrated an exceptionally significant interaction between polyoma small T antigen (PyST) and the transcription cofactor TAZ, which is comparable to the well-established interaction between PyST and protein phosphatase PP2A. These observations have led us to propose a model in which PyST recruits TAZ to proximity with PP2A for dephosphorylation and consequently protein stabilization and activation. We designed a degradable form of PyST that preserves its activity in enhancing TAZ protein abundance and promoting cell growth and survival following long-term growth factor deprivation. Gene expression profiling in PyST-expressing cells and in cells with inducible PyST protein degradation revealed gene expression alterations to a surprisingly small set of genes, suggesting that the impactful genes regulated by TAZ could be far fewer than previously considered.

Previous studies aiming to identify transcriptional targets of YAP/TAZ—as is the case for studies on the gene targets of any other transcription factors or cofactors—relied on two major strategies: gene expression profiling of cells with ectopic expression and activation YAP/TAZ (9, 26) and profiling of their DNA binding sites in the genome (10, 11, 38, 39). The binding landscape of YAP/TAZ or TEAD in the genome reveals approximately 5000 associated sites (10); however, these sites mostly reside in distal enhancer regions, posing a challenge to predicting potential genes that are regulated by YAP/TAZ. Complementing the genome loci association studies, another commonly utilized approach for studying gene expression control is to characterize gene expression alterations in cells manipulated to differentially express a given transcription (co-)factor. Earlier studies using microarrays to profile gene expression in cells with YAP or TAZ activation determined that the number of YAP/TAZ transcriptional targets ranges from ~400 to 3,000 (9, 26).

In a striking contrast, our study based on a viral antigen tool that allows temporal control of TAZ revealed a much smaller set of less than three dozen genes whose expression is significantly altered by TAZ induction or deinduction. There could be several explanations for the differences. Unlike the systems involving constitutive expression and activation of YAP/TAZ, the degradable and inducible nature of the PyST tool we identified for modulating TAZ activity likely eliminates secondary gene targets whose expression could stem from the ultimate cellular outcome of YAP/TAZ activation, such as increased cell growth, epithelial–mesenchymal transition, and acquired resistance to chemotherapeutic compounds (1, 2). A less likely explanation is that dephosphorylation at other sites on TAZ than those required for protein stabilization could affect its target/activity as a transcription cofactor.

Although we find that PyST–TAZ affinity is significant and comparable to the well-established PyST–PP2A interaction (Fig. 1 A and E), PyST has a number of other interacting proteins as revealed by our quantitative immunoprecipitation analysis. For instance, we previously focused on the interaction of PyST with TAZ paralog protein YAP and demonstrated that YAP binding is important for PyST to block the process of differentiation in C2C12 myoblasts (18). Whether PyST predominantly binds to TAZ or YAP may depend on cell type context as well as the expression levels of these proteins. Therefore, PyST may not be an extremely selective tool to modulate TAZ, other bound proteins may have critical effects either on their own or in conjunction with TAZ. Nevertheless, our study demonstrates that TAZ is potently modulated by PyST and by inducible protein degradation of PyST–FKBP12^F36V. When PyST protein stability—and consequently TAZ protein abundance—is manipulated, less than three dozen genes have significantly altered expression (Fig. 4 A and B). Thus, at least in our experimental models, the transcription targets of TAZ appear to be quite limited.

Perhaps the most important question is whether our finding is general or limited to TAZ in the context of PyST expression. This represents an important question, especially because YAP/TAZ is involved in disease pathogenesis—such as notably, the acquisition of cancer cell resistance toward KRAS inhibition (41–43). More (patho)physiologically relevant questions would include the identity of target genes of YAP/TAZ in tumor cells dependent on this pathway. There is already a hint that our conclusion has general relevance. YAP/TAZ represent attractive targets for cancer therapy, such as those driven by loss of the tumor suppressor Merlin (NF2) (44). One strategy to target YAP/TAZ is via developing small molecules to bind to the palmitate pocket of TEAD and thus blocking the interaction with YAP/TAZ. One such compound, MYF-03-69 (40), upon treating NF2-deficient mesothelioma cell line for 6 h at doses higher than its IC50 concentration, significantly reduced gene expression in only about one dozen genes (Fig. 4C). It remains to be investigated whether such an observation occurs to other YAP/TAZ/TEAD inhibitors with sufficient potency and selectivity. Nevertheless, these studies suggest that when YAP/TAZ is acutely manipulated, a very small number of genes may represent direct gene targets of the pathway and demonstrate altered gene expression.

Tumor virus antigens have been instrumental for the modern cancer research, which is likely owing to the unique characteristics of these proteins in interacting with mammalian cellular proteins and manipulating their activity. The small size of PyST offers an additional advantage for making fusion proteins to direct targeted protein dephosphorylation by PP2A. In addition, the approach utilizing inducible protein degradation might be applicable, with thoughtful protein engineering, to the study of gene expression control by other transcription factors and/or cofactors and allow for a more nuanced understanding of the transcriptional programs directed by individual factors.

Materials and Methods

Cell Culture.

MCF-10A cells were purchased from the American Type Culture Collection (ATCC). Cell identity was verified by short tandem repeat profiling (GenePrint 10 System, Promega, #B9510), performed at DFCI Molecular Diagnostics Laboratory. MCF-10A cells were cultured with DMEM/F-12 medium (Gibco, #10565018) supplemented with 5% (v/v) horse serum (Gibco, #16050122), 1% penicillin/streptomycin (Gibco, #15140163), 20 ng/mL EGF (Peprotech, #AF-100-15), 0.5 mg/mL hydrocortisone (Sigma, #H0888), 100 ng/mL cholera toxin (Sigma, #C8052), and 10 μg/mL insulin (Gibco, #12585014). Other cell lines including HEK293T and breast cancer cell lines were cultured with DMEM or RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, #10438-026). Cells were cultured in a humidified incubator with 5% CO₂ at 37 °C and passaged using Trypsin-EDTA (0.05%) (Invitrogen, #25300). Mycoplasma contamination was tested using the MycoAlert Mycoplasma Detection Kit (Lonza, #LT07-318).

Cell Growth Assays.

For short-term cell growth assays using complete medium or MCF-10A assay medium, cells were seeded in white-wall 96-well plates at the density of 1,000 cell per well. Upon control cells reaching visual confluency, cells were subjected to CellTiter-Glo assay (Promega, #G9241) according to the manufacturer’s instructions. Luminescence signal was measured by GloMax Navigator Microplate Luminometer (Promega). For long-term starvation assay, cells were seeded in 12-well plates at the density of 0.2 million cells per well. Following 2 to 3 d of culture, cells were rinsed and added with DMEM/F-12 medium that had no supplements added and was thus free of serum, any exogenous growth factor(s) or insulin. Cells were refreshed with DMEM/F-12 medium every 3 to 5 d and harvested in 4 wk or longer. Fixed cells were stained with crystal violet for visualization of cell growth. For quantification, the staining was extracted with 10% acetic acid for measurement of absorbance at 570 nm with the use of 750 nm as the reference.

Virus Packaging and Transduction.

HEK293T cells were seeded in T-25 tissue culture flasks at the density of 2.5 to 3 million cells per flask. Transfection was performed on the next day, with 12 µL 1 mg/mL polyethylenimine (PEI, Polysciences, #23966-2) and 4 µg DNA that included 2 µg lentiviral vector DNA, 1.5 µg pCMVdR8.91, and 0.5 µg pMD2-VSVG. PEI and DNA were diluted in PBS, mixed, and added to the cells following 15 min incubation at room temperature. After overnight incubation, cells were refreshed with new medium. Viral supernatant was harvested both two and 3 d after the initial transfection, filtered (0.45 µm filters), and immediately used for transducing target cells in the presence of polybrene (8 µg/mL; Millipore, #TR-1003-G). 2 d after the first transduction, infected cells were subjected to antibiotic selection until all uninfected control cells perished. The cells were then used for assays to examine the functional readouts as well as the efficiency of gene editing or ectopic expression.

Immunoprecipitation.

HEK293T were seeded in tissue culture vessels (2.5 to 3 million cells per T-25 flask, or proportionally scaled up for larger vessels), and on the next day subjected to transfection. To prepare a mixture of DNA and lipid, typically, 4 µg DNA and 12 µL PEI were diluted in PBS separately and mixed for incubation at room temperature for 15 min before being added to the cells. 24 h after transfection, cells were refreshed with serum-free DMEM, and cells were harvested following overnight incubation.

Upon harvest, cells were lysed with lysis buffer composed of 1% Triton X-100 (v/v; Sigma, #T9284) and 40 mM HEPES (pH 7.4; Gibco, #15630080) and supplemented with both protease inhibitor cocktail (Cell Signaling Technology, #5871) and phosphatase inhibitor cocktail (Cell Signaling Technology, #5870). Lysates were collected into microcentrifuge tubes, incubated on ice for 15 min with shaking, and cleared by centrifugation at 14,000 rpm for 10 min. The supernatant was transferred into new tubes for incubation with anti-Flag beads, either M2 affinity gel (Sigma M220) or anti-DYKDDDDK Tag (L5) affinity gel (BioLegend, #651501). Following overnight rotation at 4 °C, the immunoprecipitates were washed five times with lysis buffer, and after the last wash, added with 1x SDS sample buffer (50 mM Tris–HCl, pH 6.8; 2% (w/v) sodium dodecyl sulfate (SDS), 0.04% (w/v) bromophenol blue, 10% (v/v) glycerol, and 5% (v/v) β-mercaptoethanol). Boiled samples were subjected to fluorescent immunoblotting.

For quantitative proteomics analysis of proteins in the immunoprecipitates, elution buffer containing Flag peptide was used after the last wash. For elution, 30 to 50 µL solution containing 400 µg/mL Flag peptide (MedChemExpress, #HY-P0223), 50 mM Tris pH 8.0, and 150 mM NaCl was added to the beads for 15 min incubation at room temperature with frequent mixing. After centrifugation, supernatants were collected, and the beads were subjected to another round of elution. Combined supernatants were subjected to mass spectrometric analysis for protein quantification.

Antibodies, Fluorescent Immunoblotting, Quantitative Immunoprecipitation Sample Preparation, Liquid Chromatography and Tandem Mass Spectrometry, Mass Spectrometry Data Analysis, RNA Extraction and Sequencing, and RNA Sequencing Analysis.

Detailed procedures can be found in SI Appendix, Materials and Methods.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Data, Materials, and Software Availability

Bulk RNA-seq data have been deposited in Gene Expression Omnibus under Accession No. GSE232643 (45). All study data are included in the article and/or supporting information.