TEAD is required for DNA repair independent of its transcriptional function

doi:10.21203/rs.3.rs-1498950/v1

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TEAD is required for DNA repair independent of its transcriptional function

https://doi.org/10.21203/rs.3.rs-1498950/v1

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The TEA domain family members 1-4 (TEADs) are major transcription factors for YAP/TAZ transcription activators in the Hippo pathway, regulating many biological processes, including development, tissue homeostasis, and tumorigenesis through target genes. Their amplification/upregulation correlates with poor prognosis in cancer patients. Although the Hippo pathway continues to be elucidated, it is clear that TEAD largely exerts its actions via transcriptional regulation. Here, we uncover an unexpected role for TEADs in the DNA damage response. Using comparative mass spectrometry, we demonstrate that TEADs interact with several DNA repair proteins. We further show that TEADs form DNA damage-induced nuclear foci that co-localize with DNA damage markers, γH2AX, and RIF1. We also found that TEADs are required for resistance to DNA damage, maintaining genome stability, and resolution of double strand break repair that is independent from the Hippo pathway and its transcriptional role. Our results establish a new role for TEADs in DNA repair and therefore, highlight a critical consideration in therapeutically targeting the Hippo pathway.

Hippo Pathway

TEAD

DNA damage

DNA repair

Transcription factors are the gatekeepers of gene expression. Aberrant expression or regulation of several transcription factors can contribute to human diseases including cancer [1]. Among these are TEADs, which are highly conserved and ubiquitously expressed in mammals. TEADs share a common DNA binding domain (TEA domain) at the N-terminus and a transactivator binding domain at the C-terminus [2, 3]. The Hippo pathway is a highly conserved kinase cascade that maintains organ growth and tissue homeostasis [4]. Activation of this pathway is dependent on two serine/threonine kinases, mammalian STE20-like 1/2 (MST1/2) and large tumor suppressor 1/2 (LATS1/2) that phosphorylate and negatively regulate key downstream transactivators, Yes-associated protein (YAP), and its paralogue, transcriptional co-activator with PDZ-binding motif (TAZ). In the cytoplasm, phosphorylated YAP and TAZ are sequestered by binding to 14-3-3 and subsequently degraded by the proteasome facilitated by binding of β-TrCP [5]. In contrast, inactivation of the upstream kinases of the Hippo pathway leaves YAP and TAZ unphosphorylated, allowing them to translocate to the nucleus where they bind to TEADs and promote the transcription of many genes involved in cellular proliferation, homeostasis, polarity, survival, and cell fate [5].

The Hippo pathway continues to emerge as an important driver during cancer development in many tissue-types [5, 6]. A prominent example is the frequent amplification of the effector proteins, YAP, TAZ, and TEADs in squamous, head and neck, and gastrointestinal malignancies [6–8](Wang et al., 2018; Zhou et al., 2016; Sanchez-Vega et al., 2018). Additionally, induced YAP expression transforms normal epithelial cells to metastatic cells [9], and plays an important role in bypassing RAS signaling in pancreatic and colon cancer progression [10–12]. While significant progress is being made in elucidating how other signaling pathways can regulate YAP and TAZ in cancer, little is known about how TEAD is regulated independently of the Hippo pathway. Moreover, many efforts are being made to target the pathway with various approaches, including targeting TEAD. Therefore, understanding the biological impact of targeting TEAD will be critical in anticipating efficacy but also toxicity of these approaches.

DNA damage repair pathways safeguard the genome from endogenous and exogenous sources of DNA damage, including double stand breaks (DSBs) and if left unrepaired, can compromise genome integrity leading to diseases such as cancer [13]. Fortunately, these potentially detrimental aberrations to DNA are resolved by two primary repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR) [13, 14]. In NHEJ, DSBs are repaired very quickly by joining two DNA ends with very little sequence homology, an error-prone repair pathway. In contrast, HR is a multi-step process that involves extensive DNA resection around the DSB and requires sequence homology during S-phase of the cell cycle where sister chromatids are present for efficient repair.

Interestingly, YAP transcriptionally responds to DNA damage independently of the Hippo pathway. Levy et al. have shown that upon DNA damage, c-Abl kinase phosphorylates and stabilizes YAP to promote selective p73-mediated transcription of proapoptotic genes [14]. Among these proapoptotic targets is promyelocytic leukemia tumor suppressor (PML), which in turn binds to YAP and regulates its stability [15]. These findings highlight a new mechanism in which novel protein interactions and other signaling pathways can regulate the Hippo pathway effector proteins, but leaves open the question of whether TEAD functions independently of the Hippo pathway in response to DNA damage. Here, we describe a novel function of TEADs in the DNA damage response (DDR) pathway independent from the Hippo pathway and its transcriptional function. By performing protein-protein interaction studies with mass spectrometry, we found that TEADs interact with several key players in double stand break repair. We further show that TEADs form DNA damage-induced nuclear foci, and functionally demonstrate that TEADs are essential for the repair of double stand breaks, thus preventing genomic instability and conferring resistance to DNA damage. Therefore, our results uncover a novel function of the transcription factor TEADs in the DNA damage response.

TEADs bind to DNA repair proteins involved in non-homologous end joining

The identification of a functional nuclear localization signal (NLS) on TEADs or scalloped in Drosophila melanogaster led us to investigate whether we could identify novel nuclear modulators of TEADs in humans [16, 17]. Initially, we aligned the protein sequence of the NLS of all human TEAD paralogs, which revealed that it is highly conserved and overlaps with the DNA binding domain (Fig. 1a). Next, we generated doxycycline-inducible stable cell lines expressing either MYC tagged WT-TEAD3 or MYC tagged NLS-mutant TEAD3 in HEK293T and Detroit 562 (a YAP-amplified cell line). As expected, WT-TEAD3 was enriched in the chromatin fraction, while the NLS-mutant TEAD3 was predominantly enriched in the cytoplasm (Fig. 1b). Using two independent cell lines, we performed comparative mass spectrometry analysis of immuno-purified MYC tagged WT-TEAD3 or MYC tagged NLS-mutant TEAD3 to identify binding partners of TEAD3. By performing functional gene enrichment analysis, we discovered that hallmarks of E2F, G/M checkpoint, and DNA repair pathways were differently bound to WT and NLS-mutant TEAD3 (Fig. 1c). The identification of several DNA repair proteins as interacting partners, specifically interacting with WT-TEAD3 compared to the NLS-mutant TEAD3 (Fig. 1d) was surprising since TEAD primarily function during transcription. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033448.

Among the most abundant proteins that were identified in both 293T and Detroit 562 cell lines were factors involved in repairing double stranded DNA breaks including poly(ADP-ribose) polymerase 1 (PARP1), Rap1-interacting factor 1 (RIF1), X-ray repair cross-complementing protein 6 (XRCC6), and DNA-dependent protein kinase, catalytic subunit (PRKDC/DNAPKcs) (Fig. 1e). As expected, the binding affinities for TEAD canonical binding partners, YAP, TAZ, and VGLL4 were unaffected since the NLS mutation occurs near the N-terminus and not at the YAP/TAZ binding domain (Fig. 1e-f). These results were validated by performing immunoprecipitation followed by western blot analysis. Consistent with the mass spectrometry results, RIF1, XRCC6, and PARP1 specifically co-immunoprecipitated (co-IP) with WT-TEAD3, while YAP was co-immunoprecipitated with the WT- and NLS-mutant TEAD3 (Fig. 1g). Altogether, this data indicates that TEADs form a complex with DNA repair proteins, potentially indicating a role in DNA damage repair. To test this hypothesis, we performed co-IP experiments with cell lysates overexpressing WT-TEAD3 treated with bleomycin, a radiomimetic drug that induces double-stranded breaks. Interestingly, we found that RIF1, XRCC6, XRCC5, and γH2AX interaction increased binding with TEAD3 after bleomycin treatment, while that of PARP1 was unaffected (Fig. 1h).

TEADs are required for maintaining genome stability

Next, we hypothesized that if TEADs participate in DNA repair, then they might also be recruited to DNA damage sites. To address this, we generated TEADs-depleted cells using shRNAs and shNTC (non-targeting controls) in the breast cancer cell line MDA-MB-231 (Fig. 2a) and tested whether TEADs form DNA damage induced nuclear foci.

Using an antibody that detects all 4 TEADs, we found that TEADs were diffusely expressed in the nucleus of untreated cells. Surprisingly, TEADs formed nuclear foci after γ-irradiation (IR), a property commonly found in DNA repair proteins post-DNA damage (Fig. 2b). In addition, we found that TEADs co-localized with DNA damage markers, γH2AX and RIF1, supporting the hypothesis that TEADs play an important role in DNA repair (Fig. 2b). It is worth noting that depletion of TEADs by shRNA did not negatively affect the recruitment of γH2AX and RIF1, indicating the TEADs do not modulate the recruitment of these proteins to DNA damage sites (Fig. 2b). Additionally, we found that YAP or TAZ did not form DNA damage induced nuclear foci but are predominantly expressed diffusely in the nucleus (Figure S1a). Furthermore, we tested whether DNA damage-induced foci formation is independent of YAP and TAZ, and observed that TEADs formed nuclear foci that co-localized with γH2AX and RIF1 after IR in a cell line with no detectable YAP or TAZ expression (Figure S1b). Given this observation, we sought to directly test whether TEAD-mediated transcription was affected after DNA damage by utilizing a 6xGT-TEAD responsive luciferase reporter in MDA-MB-231 and Detroit 562 cells. In both cell lines, treatment with dasatinib [18], a tool compound that induces the phosphorylation and inhibits the activity of YAP/TAZ, dramatically suppressed luciferase signal over time, while treatment with IR did not (Figure S1c). Altogether, these data confirm the potential role of TEADs in DNA repair and that they act independently of YAP/TAZ and from their role in transcriptional regulation.

Next, using a comet assay [19], a gold standard in measuring the magnitude of DNA breaks, we tested if the depletion of TEADs affected the repair of DNA damage. We found that TEADs-depleted cells treated with bleomycin significantly increased DNA breaks, though TEADs depletion alone was not significantly different from the control (Fig. 2c). Moreover, the expression of γH2AX and γH2AX-ub persisted after DNA damage (γIR and bleomycin) in TEADs-depleted cells (Fig. 2d). Next, we tested chromosomal instability in TEADs-depleted cells by performing metaphase chromosome spreads. While depletion of TEADs alone did not increase chromosomal aberrations, treatment with bleomycin significantly increased chromosomal aberrations including breakages, nicks, and translocations (Fig. 2e). Altogether, these data suggest that TEADs are required to efficiently prevent the accumulation of DNA damage and to maintain genomic stability.

To test the functional significance of TEADs in DNA repair, we examined DNA damage sensitivity in TEADs-depleted cells. First, depletion of TEADs in MDA-MB-231 moderately sensitized cells as expected, and treatment with IR, bleomycin, and etoposide further sensitized these cells, suggesting that TEADs are required for the resistance to double strand break causing agents (Fig. 2f). Similar results were recapitulated upon depletion of TEADs in Detroit 562 (Figure S2a-c).

TEADs modulate DNA repair in response to double stranded breaks

Since our data strongly implicate TEADs in a complex with several important proteins involved in sensing and repairing double-stranded breaks, we next examined whether TEADs directly regulate NHEJ and/or HR using green fluorescent protein (GFP) based cell line reporters, which directly test relative repair efficiency measured by GFP positive cells. As expected, depletion of key NHEJ regulators (such as DNA ligase 4 (LIG4) and tumor suppressor P53-binding protein 1 (53BP1)) decreased NHEJ efficiency, while depletion of an HR component, BRCA2, only mildly inhibited NHEJ efficiency. Interestingly, depletion of TEADs markedly decreased NHEJ efficiency, suggesting that TEADs are involved in regulating NHEJ (Fig. 3a).

Next, we wondered whether TEADs could also regulate the cellular decision to initiate HR after double stranded breaks, therefore we performed a direct repeat-GFP assay (commonly used to access HR efficiency). We indeed observed significant inhibition of HR upon depletion of BRCA2 and not LIG4 or 53BP1. Surprisingly, depletion of TEADs also markedly inhibited HR (Fig. 3b). Since HR preferentially occurs in S-phase of the cell cycle, we tested whether the HR inhibition is simply a consequence of differing cell cycle progression in cells depleted of TEADs. Interestingly, depletion of TEADs did not change the cell cycle profile in both reporter cell lines (Figure S3a-b). These data suggest that TEADs are required for efficient NHEJ and HR. Therefore, this suggests an integral role of TEADs in the DNA damage response (Fig. 3c).

The DNA damage response is a network of processes that safeguard genomes from deleterious endogenous and exogenous DNA damage. If left unrepaired, DNA damage can produce irreversible mutations that may lead to various deleterious effects, including cancer. Here, we found that the Hippo pathway effector proteins, TEADs are directly involved in determining efficient DNA repair independent of their transcriptional function. We identified novel binding partners for TEADs by performing comparative qualitative mass spectrometry using cell lines overexpressing either WT-TEAD3 or NLS-mutant TEAD3, in which we were able to stratify unique binding partners for TEAD3 in the nucleus. Surprisingly, we discovered that proteins including PARP1, RIF1, and XRCC5/6 were enriched in the WT-TEAD3 but were absent or significantly decreased in the NLS-mutant TEAD3, while the canonical binding partners of TEADs were unaffected. It is important to note that the NLS regions of TEADs are highly conserved and therefore we believe that our results likely apply to all paralogs of TEAD. Previous studies have observed that XRCC5/6 were bound to TEAD2 or TEAD4 [6], however the biological function of this interaction was not described. Our studies provide an evidence that TEADs are important for DNA repair, and that TEADs were recruited at sites of DSBs and co-localized and interacted with DNA damage marker, γH2AX, and RIF1. Additionally, interaction with XRCC5/6 increased after DNA damage.

We further showed that TEADs are required for the efficient maintenance of genome stability and for the resistance to agents that cause double-stranded breaks. Furthermore, TEADs are also required to efficiently repair these breaks by HR and NHEJ. It is of interest to determine whether TEADs is upstream or downstream of these repair pathways, however, our data suggest that TEAD may be involved in the resolution of DNA double-strand breaks. Nevertheless, our study establishes a new unexpected role of TEADs in DNA repair in response to double-stranded breaks.

Interestingly, several members of the Hippo pathway have been shown to participate in the DNA damage response. For instance, YAP acts as a transcriptional coactivator for transcription factor, p73, which induces proapoptotic genes, suggesting that YAP can function as a tumor suppressor [14, 20]. Mechanistically, in response to DNA damage, ataxia telangiectasia mutated (ATM) phosphorylates c-Abl tyrosine kinase, and in turn, promotes the stability and binding of YAP and p73. In a parallel pathway, ATM activation by DNA damage phosphorylates the Hippo pathway activator, RASSF1A, and stimulates the activity of MST1/2 and LATS1/2 to promote YAP and p73 binding [21, 22]. This signaling pathway suggests that the Hippo pathway-YAP axis transcriptionally controls the fate of the cell in response to DNA damage. In contrast to these studies, we show that TEADs formed DNA damage-induced nuclear foci even in the absence of YAP and TAZ. We also demonstrate that YAP or TAZ did not form DNA damage-induced nuclear foci and that TEADs-mediated transcription was unchanged in response to DNA damage, suggesting that our finding is independent of YAP and TAZ, and from the transcriptional role of TEADs.

These studies, including our own, would indicate two distinct Hippo pathway-related responses to DNA damage: an indirect transcriptional output and, a direct recruitment of TEADs to DNA damage sites to modulate DNA repair. It would be interesting to understand whether there is cross talk between these pathways or a coordinated response to DNA damage.

While TEADs normally function as transcription factors, it appears that they have an additional role in regulating DNA repair after DNA damage to restore cellular homeostasis. Several studies have shown that other transcription factors play an important role in DNA damage response; for instance, several transcription factors localize to DNA damage sites, some of which are PARP1 dependent [23]. However, the exact physiological role of these transcription factors at the DNA damage sites remains unknown.

Several independent studies have begun to unravel the role of transcription factors in DNA repair. Most notably, E2F transcription factor 2 (E2F2) functions to regulate genes involved in proliferation and apoptosis, and has been implicated to directly participate in DNA repair by forming DNA damage induced foci mediated by ATM and ATR after ultraviolet (UV)-induced DNA damage [24, 25]. ATF2 is another transcription factor belonging to the AP1 family of stress responsive transcriptional activators and has been shown to localize at sites of DSBs by ATM-induced phosphorylation, however, the mechanism of how it participates in DNA repair is unknown [26]. POU domain transcription factor, BRN2 has been shown to bind to and recruited by PARP1 along with KU70 and KU80 to sites of ultraviolet B (UVB)-induced DNA damage to suppress apoptosis [27]. These studies, including our own, strongly suggest that transcription factors may play a significant role in DNA damage response and repair. More broadly, it is intriguing to speculate whether transcription factors play a dual role in general, and that this may be dependent on cellular state including growth or in this case, DNA damage.

Over the past decade, the core components of the Hippo pathway, more specifically, YAP, TAZ, and TEADs have been shown to be active players in tumorigenesis, metastasis, resistance to chemotherapies, and targeted immunotherapies [5, 10, 28]. In light of all these studies, inhibitors for the pathway are currently being developed. Our study would suggest that knockdown of TEADs partially decreases proliferation, and that treatment with γ-irradiation or chemotherapeutic agents can further sensitizes cancer cells. This indicates that a combination of a Hippo pathway inhibitor and chemotherapy may have a potential clinical benefit.

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contacts, Anwesha Dey: [email protected] and Jennie R Lill: [email protected]

Cell lines

HEK-293, Detroit 562 (pharyngeal carcinoma cells), MDA-MB-231 and SK-N-F1 (neuroblastoma cells) were cultured DMEM or RPMI supplemented with 2 mM glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, 10% heat-inactivated fetal calf serum at 37°C and 5% CO₂. All cell lines described in this study were sourced from ATCC. To generate stable lines used in our experiments, MYC/FLAG tagged- WT TEAD3 cDNA (OHu08942, GeneScript) was first subcloned into PiggyBac (pBind, Genentech) plasmid containing a puromycin and doxycycline resistance cassette by LakePharma. The WT-TEAD3 plasmid was subsequently mutated to generate R87N, R88N, R99N, R100N, K101N, V102N, and R103N (NLS mutant)-TEAD3 (LakePharma). Stable cell lines were generated by co-transfection of these plasmids using TransIT-LT1 (Mirus, MIR2300) with the transposase vector (pBO, Transposagen Biopharmaceuticals) followed by Puromycin (P9620, Millipore-Sigma) selection (2 µg/mL).

Antibodies and Chemical inhibitors

YAP (D8H1X (14074) or 1A12 (12395)), TAZ (D3I6D (70148) or E5P2N (71192)),

and Myc-tag (9B11 (2276) or 71D10 (2278) (Cell Signaling), MAX (H2) sc-8011, Santa Cruz biotechnology), panTEAD (D3F7L (13295), RIF1 (D2F2M (95558) for western blots, RIF1 (NBP2-26219, Novus biologicals) for immunofluorescence, GAPDH (D16H11 (5174)), α-Tubulin (DM1A (3873), pS139 H2A.X (05-636, Millipore-Sigma), pS139 H2A.X (20E3 (9718)), Ku70 (D10A7 (4588)) and Ku80 (2753) used for either western blotting or immunoprecipitation.

siRNAs and shRNAs

siRNAs for TEAD-1 (L-012603-00), TEAD-2 (L-012611-01), TEAD3 (L-012604), TEAD4 (L-019570-00), DNA ligase 4 (L-004254-00), 53BP1 (L-003548-00), and BRCA2 (L-003462-00) are from Horizon Discovery and use negative control siRNA (Qiagen, 1027310). MDA-MB- 231 shTEAD_1 target sequence for TEAD1: 5’-GCTCAAACACTTACCAGAGAA-3’, TEAD2: 5’-ATGACCTGTGAGATCACAAAG-3’, TEAD3: 5’-CCTGGAGTATTCAGCCTTCAT-3’ and TEAD4: 5’-GAGACAGAGTATGCTCGCTAT-3’. MDA-MD-231 shTEAD_2 contains the same target sequence from TEAD1, 3, and 4 with TEAD2 sequence: 5’-GCCTGAGCGATACATGATGAA-3’. shTEAD1/3/4 #1 sequence 5’-ATGATCAACTTCATCCACAAT-3’ and shTEAD1/3/4 #2 sequence 5’-GATCAACTTCATCCACAAGCT-3’ in Detroit 562 were cloned into PiggyBac (pBind, Genentech), previously described in Watanabe et al. 2016 (Watanabe et al., 2016).

Cellular fractionation, Western blotting, Immunoprecipitation, Mass-spectrometry

Cellular fractionation between the cytoplasm nucleus and chromatin fraction was performed using a modified protocol established by Mendez et al. [29] using fresh cell pellets resuspended in buffer A (10 mM HEPES, 10mM KCl, 1.5 mM MgCl₂, 340 mM Sucrose, 10% Glycerol, 1 mM DTT, 0.1% Triton-X, 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific)). The lysates were incubated on ice for 5 minutes. Then, the nuclear fraction was isolated by centrifugation (1300 g) for 4 mins at 4°C. The supernatant (cytoplasm) was collected, and cell pellet was washed several times with buffer A. The nuclei were lysed using buffer B (3 mM EDTA, 0.2 M EGTA, 1 mM DTT, 1X Halt™ Protease and Phosphatase Inhibitor Cocktail). The supernatant (nucleoplasm) was collected following centrifugation (1700 g) for 4 mins at 4°C. Then, the cell pellet was washed several times with buffer B. The cell pellet (chromatin) was lysed with RIPA buffer (Thermo Fisher Scientific) according to manufacturer’s instructions, supplemented with 2 mM MgCl₂, universal nuclease (Thermo Fisher Scientific), and 1X Halt™ Protease and Phosphatase Inhibitor (Thermo Fisher Scientific) Cocktail for 20 minutes on ice. Western blotting for proteins of interest were lysed using RIPA buffer according to manufacturer’s instructions supplemented with 2 mM MgCl₂, universal nuclease, and 1X Halt™ Protease and Phosphatase Inhibitor Cocktail for 20 minutes on ice. Protein concentration was measured by Bradford reagent (5000205, BioRad) using SpectraMax M5 (Molecular devices) at 595 nm. Cell lysates were run on a 4–12% Bis-Tris Plus protein gel (Thermo Fisher Scientific), and subsequently transferred onto a nitrocellulose membrane using Transblot Turbo transfer system (BioRad). The membrane was blocked, incubated with primary antibodies in 5% milk for 24 hours at 4°C. After several washes with TBST, membranes were incubated with secondary antibodies: Goat anti-mouse (926–3220, Li-cor), Goat-anti-rabbit (926-68071, Li-Cor). Images were acquired using the Odyssey Clx (Li-Cor) or by SuperSignal West Pico ECL (1856136, Thermo Fisher Scientific)

For immunoprecipitation experiments, stable expression by doxycycline treatment of MYC/FLAG WT or NLS mutant TEAD3 293T or Detroit 562 cells for 48 hours were harvested, washed with ice-cold PBS, and lysed using 20 mM Tris-HCl pH = 7.4, 150 mM NaCl, 0.5% NP40, 2 mM MgCl₂, universal nuclease (Thermo Fisher Scientific), and 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) for 20 minutes on ice. 20 mg/mL of whole cell lysates were clarified by centrifugation at 4000 x g 30 minutes at 4°C. Immunoprecipitation was carried out on cell lysates using either HA-tag or Myc-tag antibody with protein A and G Dynabeads magnetic beads (Thermo Fisher Scientific) according to manufacturer’s instructions. Immunoprecipitated proteins were eluted from the magnetic beads at 95°C for 10 minutes in 2X NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific).

In-gel reduction/alkylation and tryptic digestion

In preparation for mass spectrometry analysis, samples were separated on 4–12% Bis-Tris gel (NW04120, Thermo Fisher Scientific) under non-reduced condition. Proteins were stained with SimplyBlue stain (Invitrogen) and de-stained in water. The gel was excised from top to bottom into 14 bands per lane. Gel pieces were further de-stained in 50 mM ammonium bicarbonate (NH₄HCO₃)/30% acetonitrile (ACN) and dehydrated in 100% ACN. In-gel reduction was performed in 50 mM NH₄HCO₃ containing 50 mM of dithiothreitol (DTT) at 37°C for 1 hour, followed by a quick wash with 50 mM NH₄HCO₃ buffer. Alkylation was done using 50 mM iodoacetamide (IAA) at room temperature for 20 minutes in the dark. Excess amount of IAA was removed by washing the gel pieces with 50 mM NH₄HCO₃ /30% CAN followed by dehydration in 100% ACN. In-gel tryptic digestion was performed by hydrating the gel pieces in 10 ng/µL trypsin solution in 25 mM NH₄HCO₃ and chilled on ice for 1 hour. Excess trypsin solution was removed and digestion was performed overnight in 25 mM NH₄HCO₃ at 37°C. Peptides were extracted with 0.1% trifluoroacetic acid (TFA) in ACN. Peptides were dried to completion and re-suspended in 2% acetonitrile/0.1% formic acid/water.

LC-MS/MS analysis

Samples were re-constituted in solvent A (2% ACN/0.1% formic acid (FA)/water) and injected via an auto-sampler for separation by reverse phase chromatography on a NanoAcquity UPLC system (Waters, Dublin, CA). Peptides were loaded onto Symmetry ® C₁₈ column (1.7 mm BEH-130, 0.1 x 100 mm, Waters, Dublin, CA) with a flow rate of 1 µL/minute. A gradient of 2% solvent B to 25% solvent B (solvent A is 0.1% FA/2% ACN/water and solvent B is 0.1% FA/2% water/ACN) was applied over 35 minutes with a total analysis time of 60 minutes. Peptides were eluted directly into an Advance CaptiveSpray ionization source (Michrom BioResources/Bruker, Auburn, CA) with a spray voltage of 1.3 kV, and were analyzed using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Precursor ions were acquired in the FTMS at 60,000 resolution; MS/MS was performed in the LTQ using resonance excitation, with the instrument operated in data-dependent mode, whereby the 15 most abundant ions were selected for fragmentation in each duty cycle. For peptide identification, MS/MS spectra were searched using the search algorithm Mascot (Matrix Sciences, London, UK) against the concatenated target-decoy database (UniProtKBconcat1606) comprised of human protein sequences, known contaminants, and the reversed versions of each sequence. A 50 ppm precursor ion mass tolerance and 0.8 Da fragment ion tolerant were selected with tryptic specificity with up to 3 miscleavages. Variable modifications were permitted for methionine oxidation (+ 15.9949 Da) and IAA adduct for cysteine residues (+ 57.0215 Da), phosphorylation on serine, threonine, and tyrosine (+ 79.9663 Da), and ubiquitination on lysine residues (+ 114.0429 Da). Peptide assignments were first filtered to a 1% false discovery rate (FDR) at the peptide level and subsequently at 2% FDR at protein level. Peptide Spectral Matches (PSMs) per protein were summed per sample across all fractions from the GelC-MS experiment. The Statistical Analysis of INTeractome (SAINT) algorithm (SAINTExpress-spc v.3.6.1) [30] was run with default settings, comparing the sum of PSMs for all identified proteins enriched with MYC tagged WT-TEAD3 or MYC tagged NLS-mutant TEAD3 ,with the negative control IP, separately, per cell line. Interactions with average sum PSMs > 10, a SAINT score > 0.99, and a Bayesian False Discovery Rate (BFDR) < 0.001 were marked as significant. Significant interactions for the WT and NLS mutant TEAD3 in each cell line were submitted for an enrichment analysis against the Hallmark gene sets from MsigDB using the Broad institute’s online tool. Hallmark gene sets with a q-value < 0.05 were marked as significant. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033448.

Immunofluorescence

Cells were either cultured on 1.5 glass coverslips (cat. 64–0714, Warner instruments) or 96 well plates fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with Triton buffer (0.5% Triton X-100, 20 mM HEPES, pH 7.9, 50 mM NaCl, 3 mM MgCl₂, and 300 mM sucrose). Then, cells were washed with PBS (2x), followed by blocking using 2% BSA for 1 hour at room temperature. Primary antibodies were incubated in 0.1% Tween-20 in 2% BSA overnight, followed by 3 washes with 0.1% Tween-20/PBS. Secondary antibodies were incubated for 1 hour in 0.1% Tween-20 in 2% BSA. Cells were counter-stained with DAPI for the last 10 minutes of secondary incubation, followed by 3 quick washes with 0.1% Tween-20/PBS. Coverslips were mounted onto slides using VectaMount (H-5000, Vector Labs.)

DNA repair reporter assay

To test homologous recombination efficiency, we used pDR-GFP in U-2 OS cells, originally developed in the Jasin lab [31]. siRNAs for various targets were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 24 hours, transfection of the plasmid encoding I-SceΙ was performed with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were allowed to repair the DSBs for at least 48 hours before GFP analysis by FACS using Attune NxT Flow Cytometer. To test non-homologous recombination efficiency, we used iHN HeLa-Tet cell line (DR5001-iHNHeLa-Tet, TopoGEN) according to manufacturer’s instructions. After siRNA transfection as above, cells were treated with 2 µg/mL doxycycline to induce I-Sce1 mediated DSBs for 24 hours. Thereafter, cells were allowed to repair DSBs for at least 48 hours before GFP analysis by FACS using BDCelesta analyzer (BD Biosciences) and FlowJo software.

Comet Assay

The Comet assay was performed using OxiSelect comet assay kit (STA-351, Cell Biolabs, Inc.) following manufacturer’s instructions. MDA-MB-231 shNTC, shTEAD_1 or shTEAD_2 were treated briefly with DMSO or bleomycin for 2 hours. Cells were allowed to recover in fresh media for 24 hours. Then, cells were gently trypsinized and pelleted by centrifugation. Initially, 75 µl of molten LMAgarose was pipetted onto the comet slide and allowed to solidify at 4°C for 15 minutes. Cells were resuspended at 1 × 10⁵ cells/mL in cold PBS, followed by combining cells with molten LMAgarose at a ratio of 1:10 (v/v), and quickly pipetting 75 µL onto a comet slide, without disrupting the base gel layer. The slide was placed at 4°C in the dark for 15 minutes, then immersed in prechilled lysis buffer and kept at 4°C in the dark for 45 minutes. The comet slide was immersed in prechilled alkaline solution for 30 minutes. Then, the comet slide was transferred to a horizontal electrophoresis apparatus to perform alkaline electrophoresis at 20 V for 25 minutes. Samples were then rinsed with prechilled PBS for 5 minutes (3x). Samples were dried at room temperature in the dark, then stained with 100 µl of diluted SYBR Green I Nucleic Acid Gel Stain (S7567, Thermo Fisher Scientific Scientific) for 30 minutes. Images were captured by a confocal microscope (SP5, Leica microsystems) from four different fields and data were analyzed by CometScore.

Survival assay

50K cells were initially seeded in 6-well dishes before treating with either bleomycin, IR, or etoposide at indicated concentrations for 24 hours. Cells were then incubated with fresh media and incubated for 5–6 days. Thereafter, cells were fixed with 10% glacial acetic acid and 10% methanol for 10 minutes and stained with 0.5% crystal violet. After several rinses with ddH₂O and allowing the plates to dry, the crystal violet was resolubilized with 0.01% SDS in methanol. The absorbance was measured using SpectraMax M5 (Molecular devices) at 595 nm.

Cell Cycle Analysis

HeLa and U-2 OS cells were transfected with siControl or siTEAD Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 72 hours, cells were treated with 10 µM EdU for 15 minutes at 37°C, fixed with 4% PFA at room temperature, and permeabilized and blocked in PBS containing 10% FBS, 1% BSA, 0.1% TX-100, and 0.01% NaN₃ for 1 hour at room temperature. Click reaction was then performed following manufacturer's instructions (Thermo Fisher plus EdU cell proliferation kit). Cells were washed with PBS, re-blocked for thirty minutes, and treated with Hoechst 33342 1:10000 in PBS for 10 minutes. After washing out Hoechst, cells were imaged on OperaPhenix (PerkinElmer). Images were segmented and quantified following the protocol outlined in Chung et al [32].

Data Availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange

Submission details:

Project Name: TEAD is required for DNA repair independent of its transcriptional function

Project accession: PXD033448

Project DOI: Not applicable

Username: [email protected]

Password: klGi87hS

ACKNOWLEDGEMENTS:

The authors would like to thank all members of the Dey and Lill labs. In addition, the authors wish to acknowledge the support of the HDFCCC Laboratory for Cell Analysis Shared Resource, supported by the National Cancer Institute of the National Institutes of Health under award number P30CA082103. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

AUTHOR CONTRIBUTIONS:

This study was designed, coordinated and executed by PCC. AD and JRL, as the principal investigators, provided conceptual and technical guidance for all aspects of the project. VCP processed the protein samples and performed mass spectrometry and analysis. EV and TH analyzed all mass spectrometry data. STB performed and analyzed the HR assay and AA, as the principal investigator. BH designed and provided guidance in generating all TEAD expression and TEAD knockdown plasmids. CL performed and analyzed cell cycle experiments. MTC provided guidance in analyzing our data. NK assisted with experiments.

DECLARATION OF INTERESTS:

AA is co-founder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation; a consultant for SPARC, Bluestar, TopoRx, ProLynx, Earli, Cura, GSK; a member of the SAB of Genentech and GLAdiator; receives grant/research support from SPARC and AstraZeneca; holds patents on the use of PARP inhibitors held jointly with AstraZeneca which he has benefitted financially (and may do so in the future). All Genentech authors are employees and shareholders at Roche.

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No competing interests reported.

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TEAD is required for DNA repair independent of its transcriptional function

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Abstract

Figures

Introduction

Results

TEADs bind to DNA repair proteins involved in non-homologous end joining

TEADs are required for maintaining genome stability

TEADs modulate DNA repair in response to double stranded breaks

Discussion

Methods

Lead Contact

Cell lines

Antibodies and Chemical inhibitors

siRNAs and shRNAs

Cellular fractionation, Western blotting, Immunoprecipitation, Mass-spectrometry

In-gel reduction/alkylation and tryptic digestion

LC-MS/MS analysis

Immunofluorescence

DNA repair reporter assay

Comet Assay

Survival assay

Cell Cycle Analysis

Declarations

References

Additional Declarations

Supplementary Files

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