Hippo signaling is intrinsically regulated during progression by APC/CCdh1

Wantae Kima,b,c, Yong Suk Chod, Xiaohui Wanga, Ogyi Parke, Xueyan Maf, Hanjun Kima, Wenjian Gang, Eek-hoon Jhoh, Boksik Chai, Yun-ji Jeungb, Lei Zhangf, Bin Gaoe, Wenyi Weig, Jin Jiangd, Kyung-Sook Chungb,1, and Yingzi Yanga,1

aDepartment of Developmental Biology, Harvard Stem Cell Institute, Harvard School of Dental Medicine, Boston, MA 02215; bBiomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 34141 Daejeon, Republic of Korea; cDepartment of Biochemistry, Chungnam National University, 34134 Daejeon, Republic of Korea; dDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; eSection on Liver Biology, National Institute on Alcohol Abuse and Alcoholism, National Institute of Health, Bethesda, MD 20892; fShanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 200031 Shanghai, China; gDepartment of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; hDepartment of Life Science, University of Seoul, 02504 Seoul, South Korea; and iCardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Edited by Kun-Liang Guan, University of California, San Diego, La Jolla, CA, and accepted by Editorial Board Member Kathryn V. Anderson March 22, 2019 (received for review December 14, 2018) The Hippo-YAP/TAZ signaling pathway plays a pivotal role in plasm via interaction with 14-3-3 and subsequently degraded growth control during development and regeneration and its through β-TrCP–dependent ubiquitination (20, 21). dysregulation is widely implicated in various cancers. To further As cell proliferation is regulated by proper cell cycle pro- understand the cellular and molecular mechanisms underlying Hippo gression and Hippo-YAP/TAZ signaling is key to ensure precise signaling regulation, we have found that activities of core Hippo growth control, apart from promoting cell proliferation, Hippo- signaling components, large tumor suppressor (LATS) and YAP/TAZ signaling may also sense changes in cell proliferation YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We and tissue growth and constantly modify cell cycle progression further identified that the anaphase-promoting complex/cyclosome accordingly. YAP/TAZ are likely critical factors that bridge in- Cdh1 (APC/C) E3 complex, which plays a key role gov- trinsic and extrinsic changes with cell cycle progression, because erning eukaryotic cell cycle progression, intrinsically regulates Hippo

YAP/TAZ can be controlled by both intrinsic and extrinsic stimuli CELL BIOLOGY signaling activities. CDH1 recognizes LATS kinases to promote their that interact with MST1/2 and/or LATS1/2 kinases (1, 19, 22, 23). degradation and, hence, YAP/TAZ regulation by LATS phosphoryla- Cell cycle progression is tightly controlled by periodic expression of tion is under cell cycle control. As a result, YAP/TAZ activities peak in key components of cell cycle machinery (24). The anaphase-promoting Drosophila G1 phase. Furthermore, we show in eye and wing de- complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase velopment that Cdh1 is required in vivo to regulate the LATS homo- complex that governs cell cycle progression by regulating cyclic log Warts with a conserved mechanism. Cdh1 reduction increased degradation of key cell cycle regulators via two adaptor , Warts levels, which resulted in reduction of the eye and wing CDH1 or CDC20 (24–26). Recent reports have suggested critical sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolution- arily conserved layer of Hippo signaling regulation. Significance

LATS1/2 | YAP/TAZ | Hippo signaling | mitotic cell cycle | APC/CCdh1 The is evolutionarily conserved in the kingdom and plays essential roles in regulating tissue growth during development and regeneration. We have iden- recise growth control in embryonic development and adult Cdh1 tissue regeneration requires tightly regulated cell division and tified APC/C , a core component of cell cycle control ma- P chinery, as an evolutionarily conserved and previously unknown cell loss in response to various changes (1, 2). The Hippo signaling regulator of large tumor suppressor (LATS) kinases, which criti- pathway is evolutionarily conserved from nematodes to cally inhibit the YAP/TAZ transcription factors in transducing and plays essential roles in regulating tissue growth during de- Cdh1 – Hippo signaling. Our results suggest a model that APC/C velopment and regeneration (1, 3 5). Disruption in Hippo sig- destabilizes LATS1/2 kinases in G1 phase of the cell cycle, lead- naling leads to cancer and other devastating diseases (1, 6, 7). ing to increased YAP/TAZ activities that promote G1/S transition Originally identified in Drosophila as one required to maintain by upregulating downstream , including E2F1. precise organ sizes of the eye and wing by controlling both cell Our findings have important implications for a link between cell proliferation and survival, the Hippo signaling pathway contains proliferation and LATS-regulated YAP/TAZ activities. Hippo, Salvador, Warts, and Yorkie as core components (8–12) and receives inputs from extracellular environment as well as Author contributions: W.K., Y.S.C., X.W., X.M., L.Z., W.W., J.J., K.-S.C., and Y.Y. designed intracellular pathways to regulate a number of biological pro- research; W.K., Y.S.C., X.W., O.P., X.M., H.K., Y.-j.J., J.J., K.-S.C., and Y.Y. performed re- – search; W.K., Y.S.C., X.W., X.M., W.G., E.-h.J., B.C., L.Z., B.G., W.W., J.J., K.-S.C., and Y.Y. cesses (13 18). Central to the Hippo signaling cascade is the contributed new reagents/analytic tools; W.K., Y.S.C., X.W., O.P., X.M., H.K., W.G., E.-h.J., regulation of the Yorkie by Warts-mediated B.C., Y.-j.J., B.G., W.W., J.J., K.-S.C., and Y.Y. analyzed data; and W.K., J.J., and Y.Y. wrote . Increased Yorkie levels and nuclear the paper. localization due to Warts inactivation result in dramatic tissue The authors declare no conflict of interest. overgrowth by activating downstream gene expression to promote This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by the cell survival and proliferation. The mammalian Hippo pathway Editorial Board. consists of Hippo homologs Ste20-like MST1 and MST2, Published under the PNAS license. the scaffolding protein Salvador (SAV, also known as WW45), Data deposition: Data related to this paper have been deposited in the Gene Expression Omnibus (accession no. GSE95463). Warts homologs large tumor suppressor kinase 1/2 (LATS1/2), 1To whom correspondence may be addressed. Email: [email protected] or Yorkie homologs transcription coactivators Yes-associated pro- [email protected]. tein (YAP), and TAZ (also known as WWTR1). Activation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MST1/2 kinase inhibits YAP/TAZ by activating LATS1/2 kinases 1073/pnas.1821370116/-/DCSupplemental. (3, 19). Phosphorylated YAP/TAZ are sequestered in the cyto-

www.pnas.org/cgi/doi/10.1073/pnas.1821370116 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 roles for APC/C in various cellular processes, including genome during cell cycle (24, 32), but only CDH1 oscillated similarly to stability and tumorigenesis (27). CDH1 degrades a number of phosphorylated YAP. We next examined YAP/TAZ nuclear lo- proteins in cell cycle-dependent manner, many of which are known calization, which is regulated by phosphorylation in the DTB assay to mediate its role in negatively regulating cell proliferation and (SI Appendix, Fig. S1 A–C) and found that YAP/TAZ nuclear − − DNA replication. However, Cdh1 / mouse embryonic fibroblast localization was most pronounced in G1 phase (0 and 14 h), but (MEF) cells exhibit premature senescence and slow proliferation much reduced in G2/M phase (6 and 8 h). To further confirm that (28, 29), suggesting that some of Cdh1’stargetsmaypositively YAP/TAZ nuclear localization changes in different phases of cell regulate cell proliferation. Here, weintroduceacell-intrinsicreg- cycle under natural and asynchronous conditions, we used the ulatory mechanism in Hippo signaling by identifying LATS kinases fluorescent ubiquitination-based cell cycle indicator (FUCCI) as direct substrates of APC/CCdh1. This evolutionarily conserved system, a powerful tool for visualizing cell cycle progression in mechanism links cell cycle progression directly with Hippo signaling asynchronous cycling cells (Fig. 1B) (33). FUCCI utilizes the in growth control. phase-dependent proteolysis of the oscillators Cdt1 and Geminin. Fusion protein of Cdt1 or Geminin with the fluorescent mono- Results meric RFP (Cdt-RFP) or GFP (Geminin-GFP) serves as an in- APC/CCdh1 Is Required for YAP/TAZ Activities. The potent activity of dicator of G1 or S and G2 phase, respectively. We found that YAP/TAZ in promoting cell proliferation led us to test whether while YAP/TAZ were clearly localized in the nucleus of cells in YAP/TAZ activities are intrinsically regulated during cell cycle the G1 or G1/S phase, their nuclear localization was much re- progression. We therefore examined YAP/TAZ and Hippo sig- duced in G2 or M phase cells (Fig. 1B). These results indicate that naling activities in different phases of the cell cycle in the double YAP/TAZ activities oscillate during cell cycle and APC/C may thymidine block (DTB) assay (Fig. 1A and SI Appendix,Fig.S1A). regulate YAP/TAZ in a phosphorylation-dependent manner. We found that TAZ and phosphorylated YAP levels (pS127 by To further test whether CDH1 or CDC20 of APC/C plays a LATS kinases) (19, 20, 21, 30) oscillated during cell cycle and, in role in regulating YAP/TAZ protein levels, CDH1 or CDC20 was particular, YAP phosphorylation was reduced, while TAZ protein knocked down by two independent siRNAs in various cell lines. levels were increased in G1 and S phases (Fig. 1A and SI Ap- Reduction of CDH1, but not CDC20, led to reduced TAZ pro- pendix,Fig.S1A). Due to the high protein stability of YAP, but tein levels and increased YAP phosphorylation (Fig. 1C and SI not TAZ, TAZ is more sensitive to phosphorylation-regulated Appendix, Fig. S1D). Consistently, reduction of CDH1, but not degradation in vitro (31). The protein levels of Cyclin B, a deg- CDC20, reduced YAP/TAZ transcription activities shown by radation target of APC/C, correlated with YAP phosphorylation TEA domain (TEAD)-dependent luciferase reporter activity levels and inversely correlated with TAZ levels during cell cycle (34) (Fig. 1D and SI Appendix, Fig. S1E). Furthermore, in Cdh1- − − progression, suggesting that LATS kinase activities, and hence deficient (Cdh1 / ) MEF cells, both TAZ and YAP protein YAP/TAZ activities, are regulated during cell cycle by APC/C. levels were reduced while YAP phosphorylation was markedly The APC/C activity requires two activators, CDH1 and CDC20, to up-regulated compared with the wild-type MEF cells (Fig. 1E). ensure timely recognition and subsequent degradation of its di- Depletion of CDH1 by siRNA also significantly down-regulated verse substrates. Both CDH1 and CDC20 protein levels oscillate expression of YAP/TAZ target genes, such as CTGF and ANKRD1,

Fig. 1. APC/CCdh1 is required for YAP/TAZ activities. A B (A) HeLa cells were synchronized by double thymi- dine (2 mM) treatment and then released. Whole-cell lysates were subjected to Western blot analysis at indicated times after release. Western blot analysis was performed using the indicated antibodies. Asy, asynchronous. (B) HeLa cells were transduced with E the Premo FUCCI cell cycle sensor and incubated overnight for expression of Geminin-GFP (S/G2/M phase) and Cdt1-RFP (G1 phase). FUCCI-expressing HeLa cells were fixed with 4% fresh PFA and stained with anti-YAP/TAZ antibodies (purple). Nuclei were stained with DAPI. (Original magnification, C D 600×.) YAP/TAZ nuclear localization were quantified as percentage of cells with YAP/TAZ nuclear locali- zation in the total cells of a specific cell cycle phase. A schematic diagram of FUCCI is shown (Lower Left). (C) Asynchronized HeLa cells were transfected with control siGFP, two siCDH1s or siCDC20s. Levels of TAZ, p-YAP (S127), YAP, CDH1, CDC20, and GAPDH (loading control) were determined by Western blot analysis. (D) YAP/TAZ reporter activities (3xSd-Luc) in asynchronized cells by transfection with the indi- FG H cated plasmids in the presence of siGFP or siCDH1. n = 3 independent experiments. Error bars represent ±SD. + + − − (E) Cdh1 / WT or Cdh1 / KO MEFs lysates were subjected to Western blotting analysis with indicated antibodies. (F) Quantitative real-time PCR analysis for the expression of YAP/TAZ-target genes (CTGF and ANKRD1) after knockdown of CDH1 or YAP by siRNA in asynchronized HEK293T cells. The quantities of indicated mRNA were normalized by GAPDH. n = 3 independent experiments. (G) Western blot analysis of CTGF, p-YAP(S127), YAP, and CDH1 in nonsynchronized HeLa cells transfected with the indicated siRNAs. (H) HeLa cells were transfected with control siGFP and siAPC10. Cell lysates were subjected to Western blot analysis with indicated antibodies. In all quantified Western blotting results, repre- sentative blots are shown. Data are means ± SD of three biological replicates. **P < 0.01 (two-tailed Student’s t test). ns, not significant.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821370116 Kim et al. Downloaded by guest on September 24, 2021 without altering YAP transcription itself (Fig. 1 F and G). Con- YAP/TAZ levels, and expression of AMOTL2, CTGF,andCYR61, versely, overexpressing CDH1 enhanced TAZ-induced reporter transcription targets of YAP/TAZ (SI Appendix,Fig.S2B). Fur- activities (SI Appendix,Fig.S1F). These results indicate that CDH1 thermore, CDH1 overexpression or its depletion by small inter- is required for YAP/TAZ transcription activities. Because CDH1 is fering RNA (siRNA) decreased or increased LATS1/2 levels, known to have APC/C E3 ligase-dependent or -independent func- respectively (SI Appendix,Fig.S2C and D). Similarly, higher − − tion (35–38), we next tested whether regulation of YAP/TAZ by LATS1/2 and lower TAZ levels were found in the Cdh1 / MEF CDH1 depends on the APC/C complex. CDH1 mutants with C-box cells compared with wild-type MEF cells (Fig. 2C). These results or Fizzy domain deleted are deficient in interacting with the APC/C suggest that CDH1 regulates Hippo signaling by promoting LATS1/2 complex and they failed to enhance YAP/TAZ activities like the degradation. wild-type CDH1 (SI Appendix,Fig.S1G and H). Furthermore, we We then further tested whether LATS1/2 are previously un- found that knocking down APC10, which encodes a necessary recognized substrates of CDH1. LATS1/2 protein stability was subunit of the APC/C complex, led to down-regulation of YAP/TAZ examined by treating HeLa cells with the protein synthesis in- activities, similar to that caused by knocking down CDH1 (Fig. 1H hibitor cyclohexamide. The half-life of LATS1/2 proteins was and SI Appendix,Fig.S1I). These results indicate that CDH1 pro- significantly prolonged by CDH1 knockdown, while CDH1 over- motes YAP/TAZ activities in an APC/C-dependent manner. expression promoted LATS1/2 degradation (Fig. 2D and SI Appen- dix,Fig.S2E). Similar to the regulation of YAP/TAZ activities (Fig. APC/CCdh1 Regulates Half-Life of LATS1/2 Kinases. Our findings that 1H and SI Appendix,Fig.S1I), knocking down APC10 increased APC/CCdh1 regulates YAP phosphorylation and YAP/TAZ ac- LATS1 and LATS2 (SI Appendix,Fig.S2F), while APC/C binding- tivities prompted us to test whether LATS1 and LATS2 are sub- deficient mutants of CDH1 failed to promote LATS degradation (SI strates of APC/CCdh1, because LATS1/2 directly phosphorylate Appendix,Fig.S2G and H). APC/CCdh1 is known to induce degra- YAP/TAZ to regulate their protein levels and nuclear localiza- dation of its substrates through ubiquitin-dependent proteolysis. In- tion. LATS1/2 levels were examined during cell cycle progression deed, reduction of LATS1/2 protein levels by CDH1 was rescued by in a DTB assay and both indeed oscillated in a same pattern as treatment with MG132, a proteasome inhibitor (Fig. 2E and SI Cyclin B (Fig. 2A). In addition, LATS1/2 and CDH1 levels were Appendix,Fig.S2I). In addition, CDH1 overexpression enhanced— inversely correlated in both DTB and thymidine-nocodazole block while CDH1 knockdown reduced—poly-ubiquitination of LATS1/2 assay (Fig. 2 A and B and SI Appendix,Fig.S2A), while LATS1/ (Fig. 2 F and G and SI Appendix,Fig.S2J and K), indicating that 2 levels correlated with phosphorylated YAP levels (Fig. 2B). In APC/CCdh1 promotes LATS1/2 degradation through the ubiquitin- CELL BIOLOGY contrast, protein levels of an E3 ligase Ddb1-cullin4–associated proteasome pathway. factor 1 (DCAF1), known to promote LATS degradation (39), did To further test whether CDH1 up-regulates YAP/TAZ activ- not oscillate during the cell cycle. Neither CDC20 nor DCAF1 ities by promoting LATS1/2 degradation, we created a LATS1/2 − − inversely correlate with LATS1/2 kinase levels during the cell cycle double-knockout (DKO) (LATS1/2 / ) HeLa cell line using the (Fig. 2A). In addition, when asynchronous HeLa cells were sorted CRISPR/Cas9 technology (SI Appendix, Fig. S3). The indel into different cell cycle phases according to the DNA content, we in LATS1 and LATS2 abolished LATS1 protein ex- also found that LATS1/2, and phosphorylated YAP levels oscil- pression and resulted in a nonfunctional LATS2 truncation, re- − − lated during the cell cycle in a same pattern as Cyclin B (SI Ap- spectively (Fig. 2H and SI Appendix,Fig.S3A–C). In the LATS1/2 / pendix, Fig. S2B). Levels of LATS1/2 inversely correlated with cells, YAP phosphorylation was not detected, YAP/TAZ reporter

ABCDFig. 2. APC/CCdh1 promotes LATS1/2 kinases degrada- tion. (A) HeLa cells were first synchronized by DTB, and then released for the tindicated time. Western blot analysis was performed using the indicated anti- bodies. (B) HeLa cells were synchronized by thymidine- nocodazole block, and cell lysates were collected at the indicated time point after release. Western blot analysis was performed using the indicated antibodies. (C) + + − − Cdh1 / WT or Cdh1 / KO MEFs lysates were subjected to Western blot analysis with indicated antibodies. (D) Loss of CDH1 significantly prolongs half-life of LATS1/2. Western blotting analysis of LATS1 and LATS2 proteins E in asynchronized HeLa cells treated with cyclohexamide (CHX) for the indicated time (Upper). The line graphs show quantified LATS1 and LATS2 levels at indicated time (Lower). n = 4 independent experiments. Error bars represent ±SD, **P < 0.01 (two-tailed Student’s t test). (E) -CDH1 was transfected in asynchronized HeLa cells, which were incubated with 20 μMMG132for8h. F G HI Cell lysates were subjected to Western blot analysis with the indicated antibodies; **P < 0.01 (two-tailed Stu- dent’s t test). (F and G) HA-Ubiquitin was transfected with the indicated plasmids in the asynchronized HEK293T cells, 8 h after 20 μM MG132 treatment, LATS proteins were immunoprecipitated from the cell lysates and analyzed by Western blotting. (H) Reconstituted − − LATS1 expression in the LATS1/2 / HeLa cells rescued Hippo signaling defects. Cell lysates were subjected to Western blot analysis with indicated antibodies. (I)siGFP − − or siCDH1 was transfected into control and LATS1/2 / HeLa cells for 72 h. Cell lysates were subjected to West- ern blot analysis with indicated antibodies.

Kim et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 24, 2021 activities and target gene CYR61 expression were highly increased. (Fig. 3 A and B and SI Appendix, Fig. S5A). Furthermore, These defects in Hippo signaling were rescued by reconstituted CDH1 was found to interact with LATS1/2 in both the cytoplasm LATS1 or LATS2 expression (Fig. 2H and SI Appendix,Fig.S3D). and nucleus in the PLA assay (Fig. 3C), which was developed to − − However, the LATS1/2 / cells still exhibited a distribution in dif- detect protein interactions in close proximity in situ with higher ferent cell cycle phases, although YAP/TAZ were all localized in sensitivity and specificity compared with the traditional immu- the nucleus with no TAZ oscillation (SI Appendix,Fig.S3E and F). nofluorescent staining (43, 44). Among Hippo core components, − − Furthermore, CDH1 reductionintheLATS1/2 / cells no longer we also identified SAV1 as a binding partner of CDH1 (SI Ap- increased YAP phosphorylation, reduced TAZ levels, or YAP/ pendix, Fig. S5B), but depletion of CDH1 did not change SAV1 TAZ transcriptional activities (Fig. 2I and SI Appendix,Fig.S3G). abundance (SI Appendix,Fig.S5C). A domain-mapping study Finally, knocking down CDH1 reduced transcription activities of revealed that CDH1 and LATS interaction was mediated by the wild-type TAZ, but not the TAZ-4SA mutant that abolished WD-40 repeat domain of CDH1 (Fig. 3D). Substrate recognition phosphorylation by LATS1/2 (SI Appendix,Fig.S3H). Taken to- by CDH1 requires a variety of degradation motifs, and the most gether, these results indicate that APC/CCdh1 enhanced YAP/TAZ common ones are the destruction box (D-box) and the KEN-box activities by promoting LATS1/2 degradation. (45, 46). We found that LATS1/2 contain four potential D-boxes, Cell cycle regulation of Hippo signaling suggests that cell cycle and the third and fourth D-boxes are evolutionary conserved from arrests should lead to changes in Hippo signaling. To test this insect to human (Fig. 4A and SI Appendix,Fig.S6A). Two con- hypothesis, we induced G1 phase arrest by inhibiting CDK4/6 served D-boxes were also found in SAV1, although CDH1 did not with palbociclib (40) and partial G2/M arrest by knocking down regulate its degradation (SI Appendix,Figs.S5C and S6B). To determine whether the D-boxes are responsible for LATS deg- CDC14B (41). Because YAP/TAZ and LATS1/2 peaked in G1 – and G2/M phases (Figs. 1 and 2), respectively, indeed, we found radation, we generated individual (D1 D4) or all D-box mutants (mut-AllD) of CDH1 and tested LATS–CDH1 interaction. Un- that G1 arrest led to an increase in YAP/TAZ protein levels as expectedly, we found that all D-box mutants of LATS were still well as nuclear localization and target gene expression while able to bind to and be degraded by CDH1 (Fig. 4 B and C), LATS1/2 levels were reduced (SI Appendix, Fig. S4 A–D). Partial suggesting that additional recognition motifs are required. Further G2 arrest led to opposite changes and expected increase of domain-mapping analyses with serial LATS deletions from both β-catenin levels (SI Appendix, Fig. S4 E–H), because it has been β the N and C termini identified two crucial regions (amino acids shown that Wnt/ -catenin signaling is under cell cycle control 601–775 and 881–1130) (Fig. 4D). Because the first binding region and peaks at G2/M phase (42). Parallel FACS analysis confirmed (amino acids 601–775) contains the third (D3) and fourth (D4) D- cell cycle phase enrichment (SI Appendix, Fig. S4 B and F). boxes, we used LATS mutant constructs with D-box mutations These results further support that Hippo signaling is intrinsically and C-terminal deletion (LATSΔ881) to test whether D-box and regulated during mitotic cell cycle. another LATS domain are required for CDH1-dependent pro- Δ Cdh1 teolysis. Co-IP analysis revealed that LATS 881-mD4, but not APC/C Promotes LATS Degradation Through both Evolutionary LATSΔ881-mD3, exhibited impaired interactions with CDH1 (SI Conserved A-Box and D-Box. To investigate whether LATS1/2 are Cdh1 Appendix, Fig. S7A). As a result, its poly-ubiquitination and deg- APC/C substrates, we examined interaction of LATS1/2 with radation by CDH1 were abolished (SI Appendix,Fig.S7B and C). CDH1 by coimmunoprecipitation (co-IP) and proximity-ligation Further sequence analysis of the LATS1/2 C-terminal region assay (PLA) assays. LATS1/2 and CDH1 bound to each other (amino acids 881–1130) suggested that an evolutionary conserved A-box, a rare destruction motif recognized by CDH1, not CDC20 (47), may be critical for CDH1-regulated degradation (SI Ap- pendix, Fig. S7D). Indeed, mutating both D-box 4 and A-box, but ABCnot A-box only, impaired LATS association with CDH1 (Fig. 4 E and F) and mutant LATS with both D-box 4 and A-box mutations (mD4-mAbox) could no longer be regulated by CDH1 (Fig. 4 G and H). As a result, the CDH1-resistant LATS1 mutant showed a longer half-life compared with the wild-type control (Fig. 4I). Because the D and A boxes are located in the LATS kinase do- main where LATS activation by phosphorylation occurred, we further found that the mD4-mAbox LATS1 had lost kinase ac- D tivities as it could not be phosphorylated at T1079 (Fig. 4J)(48). These results indicate that APC/CCdh1 promotes LATS degrada- tion by binding LATS through both D-box and A-box, which are also required for LATS kinase activation.

E2F1 Is a Downstream Target Gene of YAP. Our results that YAP/ TAZ activity is up-regulated in G1 phase prompted us to test whether YAP/TAZ promotes S-phase entry. We generated Yap/Taz-deficient fl/fl fl/fl Fig. 3. LATS interacts with WD40 repeats of CDH1. (A) Flag-LATS1 was MEF cells by infecting the conditional Yap ;Taz MEF cells with transfected with or without Myc-CDH1 into the HEK293T cells. Cell lysates adenovirus carrying Cre (Ad-Cre) (SI Appendix,Fig.S8A). Loss of were subjected to co-IP and coprecipitated LATS1 or CDH1 was detected by Yap/Taz led to reduced cell proliferation, as determined by re- Western blot analysis. (B) Endogenous interaction between LATS1 and duced cell numbers or BrdU labeling (SI Appendix,Fig.S8B and CDH1 detected by co-IP followed by Western blot analysis. (C) Cells were C) and delayed G1/S transition (SI Appendix, Fig. S8D)withre- stained with rabbit anti-CDH1 antibody and/or goat anti-LATS1 antibody, duced expression of genes involved in G1/S phase transition, such and in situ interaction between LATS1 and CDH1 (red dots) was detected as , Cdc6, and E2f1 (SI Appendix, Fig. S8E). These data with secondary proximity probes as described in Materials and Methods. (Scale bars, 10 μm.) (D) WD40 repeats of CDH1 is required to interact with suggest that Hippo signaling regulates cell cycle progression and LATS1. Flag-LATS1 and the indicated Myc-Cdh1 constructs (Right) were YAP/TAZ activities peaked in G1 promotes G1/S transition. expressed in HEK293 T. Twenty-four hours posttransfection, the cells were These results are consistent with recent findings that YAP/TAZ/ pretreated with 20 μM MG132 for 8 h before collecting for co-IP and TEAD and AP-1 form a complex that synergistically activates Western blotting assays (Left). WCL, whole-cell lysates. target genes directly involved in S-Phase entry and mitosis (49).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821370116 Kim et al. Downloaded by guest on September 24, 2021 Myc-CDH1 A B C EV CDH1 (26)SSRQMLQEIRESL (D-box1) 150 (80)EIRNSLLPFANET (D-box2) Flag-LATS1 WT mAllD Human LATS1 - --++ (680)QMRKMLCQK-ESN Flag-LATS1 WT mD1 mD2 mD3 mD4 mAllD Myc-CDH1 (D-box3) 100 (753)AERDILAEADNEW (D-box4) LATS1(Flag) LATS1(Flag) MHRKHLQEIPDQS SKP2 CDH1(Myc) ** Cyclin B1 TKRAALGDLQNRG CDH1(Myc) 50 ** (EV=100) SnoN EKRLCLPQVLNSV IgG GAPDH Securin ATRKALCTV-NRA IP: ⍺-Flag 0 D-box consensus RxxLxxxxN/E/D LATS1(Flag) Relative proteins level WT mAllD AxxAxxxxN/E/D Mutant D-box CDH1(Myc) E Human LATS1 755 903 WCL 1 1130 Kinase domain Myc-CDH1 D D-box A-box Binding with LATS1 WT D-box RxxLxxxxN 1130 1130 1130 - - - CDH1 Mutant D-box AxxAxxxxN

1130 880 + - 1-1130 QR L 1- 1-775 601 881 Flag-LATS1 1- 1-600 775 WT A-box V AAAA CDH1(Myc) 1-880 + Mutant A-box IgG 1-775 + Myc-CDH1 IP: ⍺-Flag 1-600 - mA

F - CDH1(Myc) + 601-1130 - CELL BIOLOGY WT Flag-LATS1 mA mD4 + mD4 mAllD 775-1130 LATS1(Flag) + LATS1(Flag) 881-1130 CDH1(Myc) CDH1 binding regions IgG WCL IP: ⍺-Flag LATS1(Flag) WCL Flag-LATS1 G H mD4 I CDH1(Myc) mD4 Flag-LATS1 WT -mA Flag-LATS1 WT mD4-mA -/- WT mD4 mA -mA siGFP +--+-- Ctrl LATS1/2 Myc-CDH1 -+-+-+-+ siCDH1 - #1 #2 - #1#2 CHX (h) 0 3 6 9 12 036912 J

LATS1(Flag) CDH1 LATS1(Flag) - mA CDH1(Myc) LATS1(Flag) CDH1 - - mD4 mA Flag-LATS1 mD4 GAPDH GAPDH GAPDH LATS1 level snietorp evitaleR snietorp level 120 250 1.5 pLATS1 ** WT 100 mD4-mA LATS1(Flag) )001=VE( 200 ** 1.0 80 pYAP(S127) 150 * ** 60 YAP ** ** 100 0.5 40 GAPDH ** 50 20 (siGFP=100) 0.0 0 0 level Relative LATS1 036912

Relative proteins level CHX Time (hour)

Fig. 4. APC/CCdh1 requires both evolutionarily conserved A-box and D-box for LATS degradation. (A) Sequence alignment of four putative D-box motifs evolutionary conserved in LATS1 and LATS2 kinases. (B) Myc-CDH1 were transfected with either wild-type Flag- LATS1 or indicated mutant constructs, and then cell lysates were subjected to immunoprecipitation assay. (C) LATS1 mutated in all of four D-box motifs is still degraded by ectopic CDH1 expression. Flag wild-type or mutant LATS1 was transfected into HEK293T cells with or without Myc-CDH1. Cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative blots are shown; error bars represent ±SD of three biological replicates. **P < 0.01 (two-tailed Student’s t test). (D) Western blot analysis of cell lysates and immunoprecipitation derived from HEK293T cells transfected with wild-type LATS1 or truncation mutants with Myc- CDH1 construct. Twenty-four hours posttransfection, the cells were pretreated with 20 μM MG132 for 8 h before collecting (Left). Mapping studies from serial N- or C-terminal deletion reveals that LATS kinase contains two different interacting regions in CDH1. A schematic diagram showed LATS1 deletion mutants used in immunoprecipitation analysis (Right). (E) Schematic illustration of LATS wild-type and D-box, A-box LATS mutants. (F) Myc-CDH1 were transfected with either wild-type Flag-LATS1 or indicated mutant construct, and then cell lysates were subjected to immunoprecipitation assay. (G and H) Both the D-box and A-box are required for the degradation of LATS kinase by APC/CCdh1. Myc-Cdh1 or siCDH1 were transfected with wild-type Flag-LATS or indicated mutant constructs, and then subjected to Western blot analysis with indicated antibodies. Representative blots are shown; data are mean D-box and A-box signif- icantly prolonged half-life of LATS1. (I) Western blotting analysis of LATS1 proteins in HeLa cells treated with cyclohexamide (CHX) for indicated time (Upper). The line graphs show quantified LATS1 levels at indicated time (Lower). n = 3 independent experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 (two- tailed Student’s t test). (J) Mutations in LATS1 disrupted its kinase activity. LATS1 mutants with D-box and/or A-box mutations were transfected in to the LATS1/2 null mutant HeLa cells. LATS1 and YAP phosphorylation were analyzed by Western blotting.

Kim et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 24, 2021 Next, we focused on E2F1 because it is a known critical reg- of E2f1 expression in the livers of Mst1/2 DKO mice was com- ulator of G1/S transition (50) and cross-talks with the Hippo- pletely rescued by genetic removal of one allele of Yap (Fig. 5G). YAP pathway (51–54). Similar to TAZ levels, E2F1 mRNA Taken together, these results indicate that E2f1 is a direct levels have also been shown to oscillate during cell cycle (55–57). downstream target of Yap in vivo. E2F1 expression could be directly regulated by YAP/TAZ and indeed, five TEAD consensus binding sites were found in a re- Cdh1 Is Required for Organ Size Control in Drosophila Through a gion 3-kb upstream of the E2F1 transcription start site (Fig. 5A), Conserved Mechanism. Because the Hippo-Yap/Taz signaling and furthermore, these TEAD binding sites have been identified pathway has been demonstrated to control organ size in Dro- by chromatin immunoprecipitation sequencing (ChIP-Seq) as- sophila (3, 8, 9, 11, 12), we further tested whether our findings says and are associated with a transcriptionally active region as that APC/CCdh1 promotes YAP/TAZ activities by promoting determined by the enriched H3K27Ac histone binding (SI Ap- LATS kinase degradation are evolutionary conserved in vivo. pendix, Fig. S8F). By ChIP assay, we found that these TEAD Cdh1 was knocked down in the Drosophila eye imaginal discs by consensus binding sites, like the ones in the well-known target the eye-specific driver GMR-Gal4 (Fig. 6A). Consistent with a gene CTGF, were indeed occupied by YAP (Fig. 5B). Consis- role of CDH1 in promoting LATS degradation in mammalian tently, depletion of YAP/TAZ led to robust reduction of E2F1 cells, Cdh1 reduction in Drosophila dramatically increased the mRNA and E2F protein expression (Fig. 5 C and D). Because abundance of coexpressed Myc-tagged Warts (Myc-Wts), the TEAD1–4 are major DNA binding partners of YAP/TAZ, we Drosophila ortholog of LATS1/2 (Fig. 6A). Analysis of protein depleted TEAD1/3/4 by two independent siRNAs, which resul- extracted from the eye disk further confirmed increased Myc- ted in reduced expression of E2F1 and other YAP/TAZ target Wts protein levels in the Cdh1-depleted imaginal disk (Fig. genes CTGF and CYR61 (SI Appendix, Fig. S8 G and H). To 6B). In line with these results, the expression of diap1-GFP,a further confirm that YAP regulates E2f1 expression in vivo, we well-established Yorkie (the YAP/TAZ ortholog) target gene analyzed the Mst1/2 DKO livers where Mst1 and Mst2 were de- (34), was decreased by Cdh1 depletion in the eye disk (Fig. 6 C leted in hepatocytes (58). A robust up-regulation of E2f1 was and D), indicating that loss of Cdh1 led to increased Wts protein observed in vivo by RNA-seq (59), qPCR, and Western blotting levels and down-regulation of Yki transcriptional activity in analysis, while expression of other E2f families was less affected Drosophila. To further determine the functional relationship (Fig. 5 E and F). In addition, expression of both Yap/Taz and between Cdh1 and Wts, we performed a genetic interaction ex- E2f1 target genes were significantly up-regulated in the Mst1/2 periment. Suboptimal Cdh1 depletion or Wts overexpression DKO mouse livers (SI Appendix, Fig. S8I). Importantly, induction both resulted in slight reduction of eye size, but coexpression of

AB

CDE

FG

Fig. 5. YAP regulates E2F1 transcription. (A) Five putative TEAD binding elements (TBE) are located ∼3 kb upstream of the TSS of E2F1 (Upper). TEAD family transcription factors associate with the indicated motif (underlined, Lower). YAP recognizes and binds consensus sequence (GGAATG) through TEAD. (B) ChIP- qPCR assay was performed at the indicated TEAD biding sites in the promoter of E2F1, and HBB or CTGF was used as a negative or positive control for YAP binding, respectively. n = 3 independent experiments. Error bars represent ±SEM, *P < 0.05, **P < 0.01 (two-tailed Student’s t test). (C) qPCR analysis of E2F1 or YAP/TAZ-target genes (CTGF and CYR61) in cells-depleted YAP/TAZ. n = 3 independent experiments. Error bars represent ±SEM **P < 0.01 (two-tailed Student’s t test). (D) Western blot analysis of endogenous proteins of HEK293T or HeLa cells in the presence of the indicated siRNAs. (E) Heat-map of RNA-seq data showing expression of E2F family genes in indicated mice. (n = 3 mice per genotype). (F)qPCR(Left) or Western blotting analysis (Right) of E2F1 ex- pression in the livers of indicated mice. n = 3 independent experiments. Error bars represent ±SEM, **P < 0.01 (two-tailed Student’s t test). (G) Heterozygous removal of Yap in the livers of Mst1/2 DKOmice restores E2F1 expression. n = 3 independent experiments. Error bars represent ±SEM, **P < 0.01 (two-tailed Student’s t test).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821370116 Kim et al. Downloaded by guest on September 24, 2021 AC phorylated by Wts, both caused eye overgrowth. However, down- regulation of Cdh1 by Cdh1-RNAi only suppressed eye over- growth caused by overexpression of wild-type Yki, but not by mutant Yki forms (YkiS168A and Yki3SA) (Fig. 6 G and H and SI Appendix, Fig. S9A). In addition, Cdh1 knockdown did not suppress eye overgrowth caused by overexpressing a Yki-independent and constitutively activated form of Sd (Sd-GA) (SI Appendix, Fig. S9B), indicating that Cdh1 regulates eye growth through Wts BD and Yki. We also tested the effects of Cdh1 in wing imaginal discs and found that consistently, Cdh1 RNAi driven by the posterior- compartment–specific gal4 driver hh-Gal4 also led to up-regulation of GFP-Wts (Fig. 6I). Furthermore, depletion of Cdh1 in the posterior wing compartment led to size reduction compared with EFthe control wing that expressed control siRNA (Fig. 6J). Taken together, these results show that Cdh1 in Drosophila also regu- lates Hippo signaling by promoting Wts degradation in organ size control. The regulatory cascade of Cdh1-Lats-Yap/Taz that we have identified in mammalian cells also operates in an evo- lutionarily conserved way during growth control in Drosophila development. GH Discussion A key regulatory step in Hippo signaling is the regulation of YAP/TAZ phosphorylation by LATS kinases. In this study, we have identified APC/CCdh1, a core component of cell cycle control machinery, as a previously unknown regulator of LATS IJ kinases. We show with both biochemical and genetic approaches that LATS, and therefore YAP/TAZ activities, are intrinsically CELL BIOLOGY regulated during mitotic cell cycle by CDH1, an essential com- ponent of APC/CCdh1. Because CDH1 itself is also regulated by both cell cycle-intrinsic and -extrinsic factors (60), CDH1 regula- tion of Hippo signaling is likely to be important in many biological processes beyond cell cycle progression. Our results suggest a model that APC/CCdh1 destabilizes LATS1/2 kinases in G1 phase Fig. 6. Cdh1 regulates organ size via the Hippo pathway in Drosophila.(A) of cell cycle, leading to increased YAP/TAZ activities that pro- Eye discs expressing UAS-Myc-Wts and UAS-GFP (internal control) with or motes G1/S transition by up-regulating downstream gene expres- without UAS-Cdh1-RNAi under the control of GMR-Gal4 were immunos- sion including E2F1 (SI Appendix,Fig.S10). In this regard, tained with anti-GFP and anti-Myc antibodies. (B) Extracts from eye discs regulation of YAP/TAZ by LATS can sustain a positive feedback expressing UAS-Myc-Wts with or without UAS-Cdh1-RNAi under the control of GMR-Gal4 were subjected to Western blot analysis with anti-Myc anti- loop in proliferating cells by promoting cell cycle progression. Our body to detect Myc-Wts. UAS-GFP was coexpressed as an internal control. (C) findings therefore have important implications for a link between Eye discs expressing Diap1-GFP with or without UAS-Cdh1-RNAi under the cell proliferation and LATS-regulated YAP/TAZ activities. control of GMR-Gal4 were immunostained with anti-GFP and anti-Tubulin It has been shown that mitogenic growth factors can promote antibodies. (D) Extracts from eye discs Diap1-GFP with or without UAS-Cdh1- YAP/TAZ activities in a LATS-dependent way (23, 61, 62). Our RNAi under the control of GMR-Gal4 were subjected to Western blot analysis results provide a mechanism whereby mitogenic growth factors with anti-GFP and anti-Tubulin antibodies. (E) Cdh1 inhibits Wts in organ synergize with YAP/TAZ activities, by APC/CCdh1-mediated size control. Side views of a control adult fly eye (GMR > Gal4) or eyes LATS degradation through enhanced cell proliferation, which expressing UAS-Cdh1-RNAi, UAS-Wts, or both UAS-Cdh1-RNAi and UAS-Myc- promotes YAP/TAZ activation. Thus, apart from extrinsic fac- Wts under the control of GMR-Gal4.(F–H) Side views of a control adult fly > tors, intrinsic cell cycle can regulate YAP/TAZ through APC/ eye (GMR Gal4) or eyes expressing both UAS-Cdh1-RNAi and UAS-Wts- Cdh1 RNAi (F) UAS-Yki (G) or UAS-Yki3SA (H) under the control of GMR-Gal4.(I) C . Indeed, both G1 and G2 arrests altered LATS and YAP/ Wing discs expressing GFP-Wts with or without UAS-Cdh-RNAi under the TAZ activities (SI Appendix, Fig. S4 A, B, E, and F). Consistent control of hh-Gal4 were immunostained with anti-GFP and anti-Ci anti- with our observation that LATS1/2 peak at G2/M phase, LATS1 is bodies. Ci marks the anterior compartment. (J) Adult wing expressing UAS- shown to play a crucial role in controlling mitotic progression by Cdh-RNAi under the control of hh-Gal4 exhibited reduced posterior wing size. forming a regulatory complex on mitotic apparatus (63). LATS1 Eye or wing surface areas were measured by ImageJ. Error bars represent ±SD, canbindtoactinandinhibitactinpolymerization(64).Importantly, **P < 0.01 n = 4 for each genotype. (Magnification: A, C,andI,30×; E–H,10×; LATS1 regulates polymerization that affects cytokinesis × J,5 .) A, anterior wing compartment; P, posterior wing compartment. through negative modulation of LIMK1 (65). Indeed, there is evidence that LATS1/2 stringently control cytokinesis by regulat- Wts with suboptimal Cdh1-RNAi resulted in a more dramatic ing CHO1 phosphorylation and mitotic activation of LIMK1 on centrosomes (66). Finally, LATS1 has been suggested to be a reduction of eye size (Fig. 6E). Consistent with our findings in a component of the mitotic exit network in higher eukaryotes (67), cell-culture system that CDH1 regulates YAP/TAZ activities via which may explain its abilities to induce G2 arrest and regulate LATS1/2 degradation, Cdh1 knockdown did not suppress eye cytokinesis (68–70). These findings suggest that cell cycle regula- overgrowth caused by Wts knockdown, indicating that the mo- tion of LATS1/2 levels are also linked to their own functions in the lecular mechanism for CDH1-mediated LATS degradation is cell cycle, which may be independent of YAP/TAZ. evolutionarily conserved (Fig. 6F). We further tested whether Cell cycle control of proliferating eukaryotic cells involves a Yki phosphorylation by Wts is required for the effects caused by complex regulatory network. Our work provides an example and Cdh1-RNAi in vivo. Overexpression of wild-type Yki, or mutant mechanism whereby biological effects of the core engine of the forms of Yki (YkiS168A and Yki3SA) that could not be phos- mitotic cycle can be mediated by and synchronized with other

Kim et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 24, 2021 important signaling pathways, such as Hippo signaling in develop- Charlottesville, VA. The 6xMyc-tagged Cdh1 was kindly provided from ment, regeneration, and tumorigenesis. The intercommunication S. Meloche, Université de Montréal, Montreal. Myc-tagged full-length Cdh1, between Hippo signaling and cell cycle machinery indicates that Myc-Cdh1-N/F1, -N/F2, -N, and -F/WD40 were gifts from J. Lukas, University of changes in cell cycle machinery and Hippo signaling are interwoven. Copenhagen, Copenhagen. Our findings are in line with an earlier study that Wnt/β-catenin Cell Culture, siRNA Knockdown, and Transfections. HEK293T, HeLa, and signaling, also critical in growth control during development, re- Huh7 were maintained in standard conditions (DMEM supplemented with generation, and tumorigenesis, is regulated by the cell cycle and 10% FBS and 100 U/mL penicillin/streptomycin). MEF cells were cultured in peaks at the G2/M phase (42). standard conditions with 10% nonessential . siRNA transfection CDH1 has been proposed to be an oncosuppressor as it re- was performed with Lipofectamine RNAi-MAX (Life Technologies) in strains proliferation and maintains quiescent/G1 cells. Our re- antibiotic-free medium according to the manufacturer’s instructions. Oli- sults show that CDH1 in a different context can act as an gonucleotides of siRNA duplexes were purchased from Life Technologies as “,” as some of the previously unknown degradation follows: 5′- siGFP: 5-’GTTCAGCGTGTCCGGCGAG-3′, siCDH1#1: 5′-GGAA- targets of CDH1, such as LATS kinases, can inhibit proliferation. CACGCTGACAGGACA-3′, siCDH1#2: 5′-TGAGAAGTCTCCCAGTCAG-3′, siCDC20#1: Cdh1 5′-CGGCAGGACTCCGGGCCGA-3′, siCDC20#2: 5′-CGGAAGACCTGCCGTTACA-3′, It has been reported that nonperiodic activation of APC/C ′ ′ ′ leads to continuous DNA synthesis uncoupled from mitosis by siYAP: 5 -GACATCTTCTGGTCAGAGA-3 ,siLATS1:5-CACGGCAAGATAGCATGGA- 3′,siLATS2:5′-GCCACGACTTATTCTGGAA-3′. siAPC10#1: ACAAGGCATCCGTTA- transcriptionally elevating the E2F1 transcription factor, a target TATCTA, siAPC10#2: AGTACGGGAAATTGGGTCACA, siTEAD1/3/4 #1: ATGAT- of YAP/TAZ that we have identified in this study (71). In ad- CAACTTCATCCACAAG, siTEAD1/3/4 #2: GATCAACTTCATCCACAAGCT. Plasmid dition, comprehensive analyses (immunohistochemical staining transfections were carried out with PEI (Polyethylenimine; Polysciences Inc.), of tissue microarray, clustering, and statistical analysis) of more Lipofectamin 3000 (Life Technologies), or Transit-LT1 (Mirus Bio) according to the than 1,600 human benign and malignant tumors revealed that manufacturer’s instructions. CDH1 accumulates in malignant but not benign tumors (72). Therefore, tumor suppression or enhancement by the same gene Cell Cycle Synchronization. For G1/S-phase block, HeLa cells were synchronized can switch in different contexts. Related to this study, E2F1 acts as by DTB. Briefly, cells were incubated with 2 mM thymidine for 18 h followed both a tumor suppressor as well as an oncogene (73). While YAP/ by 9-h release. Cells were treated with 2 mM thymidine for another 18 h and TAZ cooperate with E2F1 to promote cell cycle progression and then released for indicated times by replacing with fresh media. For mitotic block, HeLa cells were synchronized by thymidine-nocodazole block. HeLa DNA replication (49, 53), E2F1 is not the only YAP/TAZ target cells were treated with 2 mM thymindine for 24 h. Cells were released for 3 h mediating their effects in G1/S transition and YAP/TAZ also by adding fresh media, and incubated with 100 ng/mL nocodazole for 12 h. regulates the cell cycle in E2F-independent manner (49). Cells were released from mitotic arrest for indicated times. A number of studies have reported that Hippo signaling can be regulated by various intra- or extracellular stimuli, including Cellular DNA Flow Cytomertic Analysis. The single-cell suspension (1 × 105 to cell–cell contact, mechanical stress, and growth/hormonal fac- 1 × 106 cells) was prepared in 300 μL PBS, permeabilized by cold 70% ethanol tors. Our data identify LATS kinases in Hippo signaling as in- for 30 min at 4 °C, and then washed and resuspended in 500 μL PBS, fol- trinsic factors of cell cycle machinery directly regulated by APC/ lowed by treatment with 5 μL RNase (DNase-free) at 37 °C for 30 min, chilled CCdh1, and such regulation is critical for organ size control, as on ice, and 50 μL PI (propidium iodide; Roche) treatment in the dark at room shown in the fly eye and wing. Therefore, regulation of the temperature for 1 h. DNA contents were acquired using BDCalibur and an- alyzed using the ModFit v3.3.11 software (Verity Software House). Hippo pathway by APC/CCdh1 is an evolutionarily conserved growth control mechanism. As a substrate recognition compo- BrdU Incorporation Assay. Yap/Tazfl/fl MEF cells were seeded onto four-well nent of the APC/C ubiquitin ligase complex, CDH1 has been chamber slides. After 48 h, Yap/Taz were removed by Ad-CRE infection. Ad- implicated in many cellular processes (cell cycle regulation, cell GFP infection was performed as a control. After 4 d, BrdU incorporation fate determination, and so forth) by regulating distinct substrates, assays were performed using BrdU cell proliferation (#2750; Millpore) and its role is still expanding, as exemplified by two recent studies according to the manufacturer’s protocol. showing that Cdh1/Fzr coordinates retinal differentiation with G1 arrest (74) and regulates PCP (75) through targeting Nek2 Quantitative RT-PCR Analysis. Total RNA was prepared using TRIZOL reagent kinase for degradation. Our study has revealed yet another sub- (Life Technologies) or RNAeasy mini kit (Qiagen) according to the manu- strate for CDH1: Wts/LATS1/2 kinase in the Hippo signaling facturer’s protocol. cDNA was synthesized from total RNA (1–3 μg) using pathway. Supporting our findings in mammalian cells, ablation of SuperScript II Reverse Transcriptase (Life Technologies) with random hexamer (Roche). Quantitative real-time PCR were done with SYBR Select Hippo function or overexpressing Yki in Drosophila leads to an Master Mix on StepOnePlus thermal cycler (Applied Biosystem). The increase in E2f1 level and activity (76, 77). Because D-box and A- threshold cycle (Ct) value for each gene was normalized to the Ct value for box both disrupted LATS kinase activities by abolishing GAPDH. The relative mRNA expression was calculated using ΔΔCt method. its phosphorylation at T1079 (Fig. 4J) (48), it is also possible that PCR primers for human samples were: CTGF, forward: AGGAGTGGGTGTGT- CDH1 binding to LATS itself could inactivate LATS by a con- GACGA; reverse: CCAGGCAGTTGGCTCTAATC. ANKRD1, forward: AGTA- formation change that abolishes LATS phosphorylation at T1079. GAGGAACTGGTCACTGG; reverse: TGGGCTAGAAGTGTCTTCAGAT. E2F1,forward: Furthermore, activation of LATS by phosphorylation also alters GCCACTGACTCTGCCACCATAG; reverse: CTGCCCATCCGGGACAAC. YAP,forward: the conformation of the LATS kinase domain such that they may CCTCGTTTTGCCATGAACCAG; reverse: GTTCTTGCTGTTTCAGCCGCAG. TAZ,for- be resistant to CDH1-mediated degradation. In this regard, acti- ward: GGCTGGGAGATGACCTTCAC; reverse: CTGAGTGGGGTGGTTCTGCT. vation of LATS1/2 could render them resistant to cell cycle con- GAPDH, forward: AGCCACATCGCTCAGACAC; reverse: GCCCAATACGACCAA- ATCC. Primers for mouse samples were: Ctgf, forward: CTGCCTACCGACTG- trol by CDH1. This possibility is a subject for future investigation, GAGAC; reverse: CATTGGTAACTCGGGTGGAG. Cyr61, forward: GCTCAG- which will be important to further understand how core cell cycle TCAGAAGGCAGACC; reverse: GTTCTTGGGGACACAGAGGA. E2f1, forward: machinery regulates various cellular and tissue processes through GCCCTTGACTATCACTTTGGTCTC, reverse: CCTTCCCATTTTGGTCTGCTC. Ccnd, Hippo signaling in development and regeneration. forward: AGTGCGTGCAGAAGGAGATT, reverse: CTCTTCGCACTTCTGCTCCT. Ccne, forward: GGAAAATCAGACCACCCAGA; reverse: AGGATGACGCTGCA- Materials and Methods GAAAGT. Cdc6, forward: AGTTCTGTGCCCGCAAAGTG; reverse: AGCAG- Expression Constructs. Flag-tagged full-length LATS1 or LATS2 were obtained CAAAGAGCAAACCAGG. Jag-1, forward: AGAAGTCAGAGTTCAGAGGCGTCC; from Addgene or G. Longmore (Washington University School of Medicine in reverse: AGTAGAAGGCTGTCACCAAGCAAC. St. Louis, St. Louis, respectively). Deleted forms (amino acids 1–880, 1–600, 601–1130, 775–1130, and 885–1130) and mutant versions of D-box and/or Immunofluorescence. Cells were seeded in Lab-Tek chamber slide (Thermo A-box of LATS1 were generated into p2xFlag-CMV2. Deleted form (amino Scientific), fixed for 30 min in 4% PFA in PBS, and permeablized for 20 min acids 1–775) of LATS1 was a gift from B. M. Gumbiner, University of Virginia, with 0.5% Triton X-100 in PBS at room temperature. Immunofluorescent

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821370116 Kim et al. Downloaded by guest on September 24, 2021 staining was done with standard procedures using the anti-CDH1 antibody HBB (negative control), forward: GCTTCTGACACAACTGTGTTCACTAGC; reverse: (#NBP2-15840; Novus) and anti-LATS1 antibody (#sc-9388; Santa Cruz) as CACCAACTTCATCCACGTTCACC. CTGF (positive control), forward: TGTGCCA- primary antibodies. Secondary antibodies are donkey anti-rabbit Alexa-563 and GCTTTTTCAGACG; reverse: TGAGCTGAATGGAGTCCTACACA. donkey anti-goat Alexa-488 antibodies (Life Technologies). Stained cells were mounted in DAPI mounting medium (H-1200; Vector Laboratories). Immunoprecipitation and Immunoblotting. Cells were prepared using a lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM Antibodies. Anti-YAP (4912 or 14074), anti–p-YAP (4911), anti-LATS1 (9153 or EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM so- 3477), anti-LATS2 (5888), and anti–p-Histone 3 (S10, 9701) were purchased dium orthovanadate] or RIPA buffer (Santa Cruz Biotechnology), respectively, from Cell Signaling. Anti-TAZ (560235) antibody was from BD Transduction containing protease inhibitor mixture (Roche). Immnoprecipitates or total cell Laboratories. Anti-CDH1 (34-2000) and anti-CDH1 (CC43) antibodies were lysates were analyzed by Western blotting according to standard procedures. purchased from Life Technologies and Millipore, respectively. Anti-CTGF (ab6992), anti-TEAD4 (ab58310), and anti-CDH1 (ab3242) antibodies were YAP/TAZ Immunofluorescence. HeLa cells were synchronized by DTB. Cells from Abcam. Anti-Flag (M2, F3165), anti-GAPDH (SAB1405848), anti-YAP were stained with anti-YAP/TAZ antibody (CST #8418S) overnight at 4 °C. (WH0010413M1), and anti-TAZ (T4077) antibodies were from Sigma-Aldrich. Samples were then treated with Alexa Fluor 595 Tyramide reagent (Alexa – Anti-YAP (H-125, sc-15407), anti-HA (F-7, sc-7392), anti c-Myc (9E10, sc-40), Fluor Tyramide SuperBoost Kit, B40925; Life Technologies) according to the anti-E2F1 (KH95, sc-251), anti-APC10 (B-1, sc-166790), anti-TAZ (H-70, sc- manufacturer’s protocol, and mounted with DAPI mounting medium (H- – 48805), anti p-TAZ (S89, sc-17610) anti-Cyclin B (GNS1, sc-245), anti-SKP2 1200; Vector Laboratories). Images were acquired using Zeiss 510 NLO Meta. (H-435, sc-7164), and anti-CDC20 (H-175, sc-8358) antibodies were from Santa Cruz Biotechnology. Anti-HA (11867423001) or DAPI were from Roche or Reporter Assay. YAP/TAZ transcription activities were examined in cells Vector Laboratories, respectively. Anti-mouse and rabbit HRP-conjugated seeded in 24-well plates that have been cotransfected with Sd (Scalloped, the secondary antibodies were from GE Healthcare Life Science. Anti-goat or Drosophila homolog of TEAD)-dependent luciferase reporter construct with rat HRP-conjugated secondary antibodies were from Santa Cruz and Sigma- pTK-Renilla and effector plasmids (34). The luciferase activities were ana- Aldrich, respectively. lyzed using a dual-luciferase reporter assay kit (Promega) according to the manufacturer’s instructions. In knockdown experiments, cells were first In Situ PLA. In situ PLA experiments were performed as described previously. transfected with 20 nM siRNA for 48 h before luciferase assay. Primary antibodies (rabbit α-CDH1 and Goat α-LATS1 in blocking solution) was incubated at room temperature for 2 h. Cells were washed for five times Fly Stocks, Transgenes, Immunostaining, and Western Blot. Transgenic lines for 5 min in PBS plus 0.1% Tween 20. To detect protein–protein interaction used were: UAS-Cdh1-RNAi (VDRC #25553 and #25550); wtsMI05605-GFSTF.0,a between LATS1 and CDH1, secondary proximity probes (Rabbit-PLUS and protein trap insertion transgenic line expressing GFP-Wts (Bloomington Goat-MINUS) (Olink Biosciences) were incubated for 90 min at 37 °C. Cells

Stock #56808); Diap-GFP (34), UAS-Myc-Wts (9), GMR-GAl4, UAS-Yki, UAS- CELL BIOLOGY were washed five times for 5 min in 10 mM Tris·HCl (pH 7.5) plus 0.1% YkiS168A and UAS-SD-GA (34), and UAS-Yki3SA (78). The antibodies used for Tween 20 at 37 °C, then twice for 5 min in PBS plus 0.1%Tween 20. All immunostaining and Western blot analysis were rabbit anti-GFP (Molecular subsequent steps were performed according to the Duolink proximity liga- Probes), mouse anti-Myc (Santa Cruz), rat anti-Ci, 2A1 (DSHB), and Cy2-, Cy3- tion assay detection kit protocol (Olink Biosciences). Wide-field images were conjugated secondary antibodies (Jackson Immuno Research Laboratories). Im- collected using a Personal DeltaVision system (GE Healthcare) mounted on ages were captured by Zeiss LSM710 confocal microscopy. For protein extract of an inverted Olympus IX71 microscope with a Plan Apo 60×/1.42 oil objective eye discs, eye imaginal discs were collected from the late third-instar larvae and lens. All images were acquired using a pco.edge sCMOS camera with 1 × 1 lysed in Nonidet P-40 cell lysis buffer with protease inhibitor mixture (Roche). binning and a 1,024-pixels × 1,024-pixels imaging field. PLA excitation Lysates were cleared by centrifugation and then subjected to SDS/PAGE. (100%) and DAPI excitation (10%) were collected in emission filters 632/50 and 435/48, respectively. The z-stacks were collected (21 images per stack with a z-interval of 0.1 μ) for each image. All images were deconvolved using Statistical Analysis. All experiments were performed at least three in- an iterative constrained method with 10 cycles, medium noise filtering. dependent times (unless noted otherwise) and representative data are Maximum-intensity projections were created for each deconvolved stack. All shown. Statistical analysis between groups was performed by two-tailed ’ image capture and postprocessing was done using GE’s SoftWoRx software Student s t test in Graphpad Prism 7 to determine significance when only ’ package v6.0.0. two groups were compared. One-way ANOVA with Tukey s post hoc tests were used to compare differences between multiple groups. P values of less ChIP Assay. ChIP was performed by using a modified protocol from the ChIP than 0.05 and 0.01 were considered significant. Error bars on all graphs are Assay Kt (17–295; Millipore). Briefly, cells were cross-linked in 1% formal- SD unless otherwise indicated. dehyde for 15 min at room temperature, and 0.125 M glycine was added to fixed cells to stop the reaction. Cells were washed, harvested with cold PBS, ACKNOWLEDGMENTS. We thank members of the Y.Y. laboratory for stimu- and resuspended in lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris at lating discussion; S. Wincovitch (NIH/National Research Institute) for pictures of proximity-ligation assay; S. Anderson (NIH/National Human pH8.1). Nuclei were disrupted by sonication (Misonix XL-2000) by performing × Genome Research Institute) for flow cytometry; J. Lukas (Institute of Cancer eight rounds of 3 5-s pulses with at least 20-s rest between pulses and 2- Biology, Denmark) for reagents; and Radhika Khetani and Michael Steinbaugh – min rest between rounds to obtain fragments of average 200 1,000 bp in of the Harvard Chan Bioinformatics Core from the Harvard T. H. Chan School of size. The sonicated cell supernatant was diluted in ChIP dilution buffer Public Health for assistance with the Gene Expression Omnibus submission. The (0.01% SDS, 1.1%Triton X-100, 1.2 mM EDTA, 20 mM Tris at pH8.1 and assistance of Radhika Khetani and Michael Steinbaugh was supported by fund- 167 mM NaCl). Suitable amounts of chromatin were immunopreciptated ing from Harvard Catalyst j The Harvard Clinical and Translational Science Center with specific antibodies overnight. Antibodies used were IgG (#026102; Life (NIH Award UL1 RR 025758 and financial contributions from participating insti- Technologies) and YAP (#14074; Cell Signaling). complexes were recovered tutions). This study is supported by National Human Genome Research Institute on ChIP-grade Protein A/G plus agarose bead (#26195; Life Technologies). intramural research grants, and NIH Grant AA025725, R01CA222571 (to Y.Y. and X.W.); NIH Grant GM118063 and Welch Foundation Grant I-1603 (to J.J.); NIH qPCR was performed using primer sets flanking the predicted TEAD binding Grant 1GM089763 (to W.W.); a National Institute of Alcohol Abuse and Alcohol- sites. Primer sequences used are as follows: TBE1, forward: GCCCAGT- ism intramural research grant (to B.G.); National Research Foundation of Korea GACTGTGGATTTT; reverse: GTCCTGCTGAAGCAGAAAGG. TBE2/3, forward: Grant NRF-2016R1A5A1010764 (to E.-h.J.); the Basic Research Program through GTGTTACTGGGACCCTGTGG; reverse: CCCATCCCCTCTCCATAAAG. TBE4, for- the National Research Foundation Grant NRF-2018R1C1B6002749 (to W.K.); and ward: GGACCTACCCCTCAGCTTCT; reverse: AGAAGGGACCACAGAGACCA. the Korea Research Institute of Bioscience and Biotechnology Initiative of the TBE5, forward: CCCCTGTCTACTGGACTGTGA; reverse: CTGCAAGTCCCATTTTAGCC. Korea Research Council of Fundamental Science and Technology (K.-S.C.).

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