Fine-Tuned and Cell-Cycle-Restricted Expression of Fusogenic Protein Syncytin-2 Maintains Functional Placental Syncytia

Report

Fine-Tuned and Cell-Cycle-Restricted Expression of Fusogenic Protein Syncytin-2 Maintains Functional Placental Syncytia Graphical Abstract

Authors Xiaoyin Lu, Rui Wang, Cheng Zhu, ..., Yan Gu, James C. Cross, Hongmei Wang

Correspondence [email protected] (J.C.C.), [email protected] (H.W.)

In Brief Lu et al. demonstrate that G0-phaserestricted fusogenic protein Syncytin-2 is essential for maintenance of functional human placental syncytia. Overexpression of Syncytin-2 overrides cell-cycle restriction and results in functionally compromised syncytia carrying mitotic features. p21 coordinates with transcription factor GCM1 to regulate Syncytin-2 transcription to guarantee appropriate human trophoblast fusion.

Highlights d

The expression of fusogenic protein Syncytin-2 is G0 phase restricted

d

Overexpression of Syncytin-2 leads to fused trophoblast cells with mitotic features

d

Syncytin-2-induced syncytia exhibit compromised function

d

p21, together with GCM1, regulates Syncytin-2 transcription

Lu et al., 2017, Cell Reports 21, 1150–1159 October 31, 2017 ª 2017 The Authors. https://doi.org/10.1016/j.celrep.2017.10.019

Cell Reports

Report Fine-Tuned and Cell-Cycle-Restricted Expression of Fusogenic Protein Syncytin-2 Maintains Functional Placental Syncytia Xiaoyin Lu,1,7 Rui Wang,1,7 Cheng Zhu,1 Haibin Wang,1 Hai-Yan Lin,1 Yan Gu,2 James C. Cross,3,4,5,6,8,* and Hongmei Wang1,8,9,* 1State

Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China Second Hospital of Tianjin Medical University, Tianjin 300211, China 3Department of Biochemistry and Molecular Biology 4Department of Comparative Biology and Experimental Medicine 5Department of Obstetrics and Gynecology 6Department of Medical Genetics University of Calgary, Calgary, AB T1S 1A2, Canada 7These authors contributed equally 8Senior author 9Lead Contact *Correspondence: [email protected] (J.C.C.), [email protected] (H.W.) https://doi.org/10.1016/j.celrep.2017.10.019 2The

SUMMARY

Many types of multinucleated cells (syncytia) generated by cell-cell fusion are post-mitotic, but it remains unclear how this state is maintained and why. Here, we utilized the fluorescent ubiquitination-based cell-cycle indicator (Fucci) reporter system to show that human placental trophoblast cells were all in the G0 phase before they fuse. Expression of the fusogenic protein (fusogen) Syncytin-2 was confined to G0 cells. Overexpression of Syncytin-2 in cycling cells overrode the cell-cycle restriction and enabled fusion of cells in the S/G2/M phases but resulted in the unstable syncytia retaining mitotic features. The Syncytin-2-induced syncytia were functionally compromised with respect to pathogen defense and hormone secretion. We found that, during trophoblast fusion, the cell-cycle inhibitor p21 interacted with the GCM1 transcription factor, and this complex bound to the promoter of Syncytin-2 and promoted its transcription. These findings demonstrate that G0-restricted Syncytin-2 expression is a prerequisite for development of functional postmitotic syncytia. INTRODUCTION Cell-cell fusion occurs in diverse types of cells and organs in different species such as mating in yeast, giant cell sculpting in nematodes, fertilization, bone formation, myogenesis, and placentation (Aguilar et al., 2013). The continuous layer of human syncytiotrophoblast generates a surface area of 12–14 m2 at the feto-maternal interface in the term placenta (Burton and Jauniaux, 1995) and functions as a barrier against pathogens such

as Listeria monocytogenes (Robbins et al., 2010), a producer of hormones such as progesterone, and a unit where gas and nutrient exchange occurs (Cross et al., 1994). The number of nuclei in the syncytiotrophoblast increases from approximately 0.6 billion at 13–15 weeks of gestation to 5.8 billion at 37–39 weeks of gestation (Simpson et al., 1992), indicating a continuous fusion process. The expanding syncytiotrophoblast is both dynamic and stable, corresponding to the active fusion process and the structurally unaltered single unit, respectively. To generate syncytia, fusogenic proteins (fusogens) bridge plasma membranes and directly merge cells into multinucleated units. To date, only two families of cell-cell fusogens have been identified: F proteins (EFF-1 and AFF-1) in nematodes and syncytins in mammals (Podbilewicz, 2014). Syncytins were captured from retroviruses during evolution and are predominantly expressed in the placenta (Aguilar et al., 2013). They have been identified across different species including simians, muroids, leporids, carnivores, and ruminants (Esnault et al., 2013), such as Syncytin-A and -B in mice, and Syncytin-1 and -2 in humans. Syncytin-1 and -2 are involved in the formation of multinucleated syncytiotrophoblast from mononucleated cytotrophoblast cells (Aguilar et al., 2013). The syncytia generated by fusion under physiological conditions are thought to be primarily post-mitotic. This unique cellcycle property is also viewed as a sign of terminal differentiation (Schneider et al., 1994). It has been shown that the syncytiotrophoblast remains post-mitotic (Richart, 1961). Therefore, it has been assumed that mononucleated cells must exit the cell cycle before undergoing fusion. However, direct evidence about whether cells leave the cell cycle before they fuse, and whether cell-cycle status is directly associated with fusogen regulation and cell fusion abilities are still lacking due to technical limitations of real-time cell-cycle monitoring and tracing. Furthermore, whether the post-mitotic feature of the syncytiotrophoblast cells contributes to their stable nature and function is an open question.

1150 Cell Reports 21, 1150–1159, October 31, 2017 ª 2017 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

In this study, we took advantage of the fluorescent ubiquitination-based cell-cycle indicator (Fucci) reporters (SakaueSawano et al., 2008) to track cell-cycle status during the dynamic cell-cell fusion process using time-lapse microscopy. We observed that only G0-arrested cells were able to fuse due to the restricted expression of Syncytin-2. Overexpression of Syncytin-2 in cycling cells resulted in the formation of syncytia, but they were unstable and had functional defects. Interestingly, Syncytin-2 expression was detected in proliferative cytotrophoblast cells in the placenta in some cases of recurrent spontaneous abortion (RSA). We found that the cell-cycle inhibitor p21 interacted with transcription factor GCM1 to bind the promoter region and promote transcription of the Syncytin-2 gene. These results directly linked expression of fusogens with the cell-cycle machinery, which was essential for the formation and maintenance of functional syncytia. RESULTS Human Trophoblast Cells Are in G0 Phase before They Fuse Previous studies indicated that human multinucleated syncytiotrophoblast cells lack DNA synthesis (Richart, 1961). To confirm this finding, human placental villi were collected at 7 weeks of gestation, and immunofluorescence was performed to detect the expression of the cell-cycle marker Ki67. Ki67 staining persists in active phases of the cell cycle (G1, S, G2, and M), whereas it is lost in post-mitotic G0 cells (Yerushalmi et al., 2010). Ki67positive signals were detected in some of the cytotrophoblast cells, while they were completely absent from the syncytiotrophoblast (Figure 1A). In a second model, human BeWo trophoblast cells were treated with forskolin to induce fusion (Wang et al., 2014). The syncytium is characterized by multiple nuclei within a cell boundary marked by E-cadherin immunostaining. To make sure that multinucleated cells produced by BeWo cell fusion represented human syncytiotrophoblast rather than placental bed trophoblast giant cells, we performed immunostaining and western blotting for HLA-G, a marker for extravillous trophoblast including trophoblast giant cells. HLA-G was not detectable in the fused BeWo cells (Figure S1A; Figure 1B). We next checked the proliferation ability of these cells. No immunostaining of Ki67 was detected in the multinucleated syncytium (Figure 1B, arrowhead), and Ki67-positive signals were only detected in some mononucleated cells (Figure 1B, arrows). To trace the highly dynamic cell-cycle changes, we took advantage of the Fucci reporter system (Sakaue-Sawano et al., 2008), which labels individual cells in the G1/G0 phase with red fluorescence and those in the S/G2/M phases with green. Modifications were made to the Fucci2 probes (Abe et al., 2013), which consist of human Geminin (amino acids 1–110) fused to mVenus and human Cdt1 (amino acids 30–120) fused to mCherry, by replacing mVenus with the brighter and more photostable mNeonGreen (mNG) (Shaner et al., 2013) (Figure S1C). This modified reporter was named ‘‘FucciNG’’ (Figure 1C). BeWo cells carrying FucciNG (hereinafter referred to as BeWoFucciNG) were generated by lentiviral infection (Figure S1D). BeWo-FucciNG cells were cultured and subjected to time-lapse microscopy. The expected transition from red to green fluores-

cence during the progression of the cell cycle from G1 to S phase was observed (Figure S1E). The fluorescent pattern throughout the entire cell cycle was as follows: no color (right after mitosis) to red (G1) to yellow (G1/S) to green (S/G2/M) (Figure S1E), indicating the efficacy of using the FucciNG reporter to trace cell-cycle progression in placental trophoblast cells. To observe syncytium formation in live cells, BeWo-FucciNG cells were transfected with a blue fluorescent protein (mTagBFP2) or an infrared fluorescent protein with a nuclear localization signal (NLS-iRFP670, magenta) (Shcherbakova and Verkhusha, 2013). These two populations of cells were mixed and treated with forskolin to induce syncytialization (Figure 1D). As revealed by confocal microscopy, all of the nuclei in the fused BeWo-FucciNG cells that were double-positive for mTagBFP2 and NLS-iRFP670 were always red and never green (Figure 1E), indicating that they were in the G1 or G0 phase. Time-lapse imaging showed that only red cells (G1/G0 phase) fused to form a syncytium (Figure 1F; Movie S1). Combined with the fact that nuclei in the syncytiotrophoblast in vivo are Ki67-negative (G0 phase), we concluded that trophoblast cells exit the cell cycle and enter G0 before fusing. To further confirm these results, a dye dilution assay was performed using carboxyfluorescein succinimidyl ester (CFSE), a stable fluorescent cell staining dye (Figure 1G). BeWo cells were labeled with CFSE, treated with forskolin, and sorted into two populations: CFSE-dim (more proliferative) and CFSE-bright (less proliferative). These two populations of cells were then treated with forskolin, and CFSE-dim cells exhibited much lower fusion index than did CFSE-bright cells (Figure 1H). The Fusogenic Protein Syncytin-2 Is Restricted in the G0 Phase Fusogens are the fundamental driving forces of cell-cell fusion. During forskolin-induced fusion of BeWo-FucciNG cells, expression of Syncytin-2 was significantly elevated, accompanied by an increase in the secretion of the hormone human chorionic gonadotropin (hCG), whereas expression of Syncytin-1 remained unchanged (Figure 2A), which highlights the role of Syncytin-2 in promoting cell-cell fusion (Blaise et al., 2003). To compare the level of Syncytin-2 between G1/G0 cells and cells in other cell-cycle phases, forskolin-treated BeWo-FucciNG cells were sorted into green (representing the S/G2/M phases) and red (representing the G1/G0 phase) populations. Western blots revealed that Syncytin-2 was highly enriched in the G1/ G0 fraction but was barely detectable in the S/G2/M population (Figure 2B). To test whether this observation was also true in vivo, immunofluorescent staining for Syncytin-2 and the proliferation marker Ki67 was performed on human placental tissues in the first trimester. Syncytin-2-positive signals were only detected in the cytotrophoblast cells that were Ki67-negative (Figure 2C), indicating that only cells arrested in the G0 phase expressed Syncytin-2. Ectopic Expression of Syncytin-2 Promotes Fusion of Proliferative Cells but Results in Unstable Multinucleated Cells Because expression of Syncytin-2 was detected only in G0 cells and only G0 cells were able to fuse, we next investigated whether

Cell Reports 21, 1150–1159, October 31, 2017 1151

Figure 1. Trophoblast Cell Fusion Is Accompanied by G0 Arrest (A) Left: immunofluorescence of Ki67 in human placental villi at 7 weeks of gestation from the first trimester. CTB, cytotrophoblast cells; STB, syncytiotrophoblast. Scale bar represents 50 mm. Right: schematic representation of a cross-section through a human placental villus from the first-trimester placenta. Arrows indicate a layer of mononucleated CTB and a layer of multinucleated STB. (B) Immunofluorescence staining of Ki67 and E-cadherin in BeWo cells treated with forskolin. Nuclei were stained with DAPI (blue). Scale bar represents 20 mm. (C) Overview of the FucciNG system. (D) A schematic overview of the experimental procedure. FSK, forskolin. (E) Confocal images of cells treated as shown in (D). Scale bar represents 50 mm. (F) Time-lapse imaging of cells treated as shown in (D), except that only mTagBFP2 was transfected. Still images from representative Movie S1 are shown; 0 hr corresponds to 03:59:59. Arrows indicate fusion (mTagBFP2-labeled blue cytoplasm flow). Scale bar represents 20 mm. (G) Dye dilution assay for cell proliferation using CFSE. CFSE is a stable fluorescent cell staining dye that becomes diluted after mitosis. CFSE-bright cells indicate slow proliferation. (H) Left panel: immunofluorescence with E-cadherin antibody. Nuclei were stained with DAPI (blue). Right panel: the fusion index of BeWo cells after forskolin treatment. Data are shown as mean ± SD of three experiments (**p < 0.01). White dotted line circled area indicates the syncytia. Scale bar represents 20 mm. See also Figure S1 and Movie S1.

1152 Cell Reports 21, 1150–1159, October 31, 2017

Figure 2. The Fusogenic Protein Syncytin-2 Is Highly Enriched in the G0 Phase, while Forced Expression of Syncytin-2 in Cycling BeWo Cells Enables the Fusion of S/G2/M Cells, which Leads to Unstable Syncytia with Mitotic-like Features (A) Western blot of BeWo-FucciNG cells treated with/without forskolin (FSK). b-Actin was a loading control in all experiments. (B) A schematic overview of the experimental procedure (left) and the western blot of the sorted cells (right). Note that Syncytin-2 is highly enriched in the red fraction. (C) Localization of Syncytin-2 and Ki67 in human placental villi from 7 weeks of gestation by immunofluorescence. The area within the white dotted lines indicates the cytotrophoblast layer. The arrowhead indicates a cell that expressed Syncytin-2. Scale bar represents 50 mm. (D) A schematic overview of the experimental procedure. (E) Confocal images of cells treated as shown in (D). Scale bar represents 20 mm. (F) Time-lapse imaging of cells treated as shown in (D), except that only mTagBFP2 and Syncytin-2 were transfected. Still images from representative Movie S2 are shown; 0 hr corresponds to 09:19:55. Arrows indicate where fusion occurred (mTagBFP2-labeled blue cytoplasm flow). Note that cells about to fuse contained green nuclei. Scale bar represents 50 mm. (G) Time-lapse imaging of Syncytin-2-induced fused BeWo cells transfected with H2B-mCherry and mNG-farnesyl. Still images from representative Movie S4 are shown. 0 hr corresponds to 05:50:03. Arrows indicate a syncytium that underwent mitosis. Scale bar represents 20 mm. See also Figure S2 and Movies S2, S3, and S4.

Cell Reports 21, 1150–1159, October 31, 2017 1153

introduction of Syncytin-2 into cells in the S, G2, and M phases would allow these cells to fuse. Syncytin-2 was ectopically expressed in BeWo-FucciNG cells along with the mTagBFP2 or NLS-iRFP670 cell markers (Figure 2D). Interestingly, green nuclei (S/G2/M phase cells) were detected in the multinucleated syncytia (Figure 2E). A time-lapse imaging study on BeWo-FucciNG cells transfected with Syncytin-2, together with mTagBFP2 (to visualize the cytoplasm flow), further revealed that green cells indeed underwent fusion (Figure 2F; Movie S2). To determine the fate of Syncytin-2-driven syncytia, a Cre/ loxP-based cell-cell fusion reporter system (Figure S2A) was established in 293T cells. This system allowed us to trace the genuinely fused cells (GFP-positive). Syncytin-2 was introduced into this system, and the whole process of cell fusion was observed. Strikingly, the Syncytin-2-induced fused 293T cells were unstable and tried to divide (Figure S2B, arrows; Movie S3). To verify this phenomenon in trophoblast cells and to better visualize chromosomes, we transfected BeWo cells with H2B-mCherry and plasma membrane-localized mNG and subjected them to time-lapse imaging. Chromosomal characteristics of metaphase, anaphase cells, and cytokinesis were observed in the fused BeWo cells (Figure 2G; Movie S4). Taken together, these results indicated that cells at cell-cycle stages other than G1/G0 could fuse when Syncytin-2 was ectopically expressed, but fusion was not accompanied by cell-cycle exit. Syncytin-2-Induced Syncytia Are Functionally Compromised Syncytin-2-induced fusion of cells in cell-cycle phases other than G0 led to the formation of unstable syncytia, and so we wanted to know if they were also functionally defective. Human syncytiotrophoblast is resistant to infection by pathogens such as Listeria monocytogenes (Robbins et al., 2010). Forskolininduced or Syncytin-2-induced BeWo cells were exposed to Listeria monocytogenes in culture, and the ability of the bacteria to invade the cells was examined by immunofluorescence. Forskolin-induced, post-mitotic syncytia exhibited more resistance to Listeria monocytogenes invasion than the neighboring mononucleated cells (Figure 3A, left panel, white dotted line circled area). By contrast, Syncytin-2-induced syncytia exhibited much less resistance and contained abundant bacteria similar to neighboring unfused, mononucleated cells (Figure 3A, right panel). To further confirm that we were observing genuine fused cells, a modified Cre/loxP reporter system (where fused cells turn into green) in BeWo cells was used (Figure S3A). Synytin-2induced syncytia were less resistant to Listeria monocytogenes, compared to forskolin-induced syncytia (Figure S3B). The production of progesterone, the key hormone to maintain pregnancy, is another essential aspect of syncytiotrophoblast function. Forskolin-induced cells produced more progesterone than control cells, whereas Syncytin-2-induced cells produced progesterone at levels similar to control cells (Figure 3B). The enzyme CYP11A1 (cytochrome P450 family 11 subfamily A member 1) that is involved in progesterone synthesis was significantly upregulated in forskolin-induced fusion but not in Syncytin-2induced fusion (Figure 3D). Mitochondrial fragmentation is essential for progesterone production (Wasilewski et al., 2012), and so

1154 Cell Reports 21, 1150–1159, October 31, 2017

we examined mitochondrial morphology in forskolin-induced compared to Syncytin-2-induced syncytia. BeWo cells carrying a Cre/loxP-based fusion reporter system were transiently transfected to express Syncytin-2. Compared to forskolin-induced syncytia, whose mitochondria were fragmented, Syncytin-2induced syncytia contained many elongated mitochondria (Figure 3C). Similar results were observed in non-modified BeWo cells transfected with Syncytin-2 (Figure S3C). We further examined the expression of mitochondria fragmentation-related proteins. During forskolin-induced fusion, expression of Drp-1, a mitochondria fragmentation-promoting protein, was increased while Opa-1, a mitochondria fragmentation-inhibiting protein, was decreased. However, during Syncytin-2-induced fusion, Drp-1 and Opa-1 exhibited no obvious changes (Figure 3D). Collectively, these data indicate that Syncytin-2 expression alone can induce fusion but the syncytia have structural and functional defects. The fact that overexpression of Syncytin-2 in cycling cells resulted in structurally and functionally compromised syncytia prompted us to investigate whether this mechanism had clinical relevance. Since these abnormal syncytia secreted less progesterone, we focused on pregnancy-related diseases associated with relatively lower serum progesterone concentrations including spontaneous abortion (Hahlin et al., 1990). We collected placental samples from recurrent spontaneous abortions and performed immunostaining for Ki67 and Syncytin-2, to determine whether abnormal fusion occurred in these tissues. In contrast to gestation age-matched controls where Ki67 was never detected in Syncytin-2-postive cytotrophoblast cells (scanned over 5,000 cells from each of n = 8 placentas), co-localization of Ki67 and Syncytin-2 was detected in cytotrophoblast cells in 2 cases out of 12 recurrent spontaneous abortionaffected placentas (Figure 3E). The Syncytin-2/Ki67 double-positive cells in the two cases tended to be clustered rather than widely distributed in the villous cytotrophoblast population and represented less than 20% of all Syncytin-2-positive cells. These data indicate that expression of Syncytin-2 in cycling cells can occur, but only in pathological conditions. The Cell-Cycle Regulator p21 and Transcription Factor GCM1 Regulate Syncytin-2 Transcription The above observations indicated that G0 arrest was a prerequisite for Syncytin-2 expression in cytotrophoblast cells and their ability to develop into structurally and functionally normal syncytiotrophoblast. It was therefore conceivable that cell-cycle inhibitors that arrest cells in G0 phase may be involved in Syncytin-2 induction. A previous report demonstrated that the cyclindependent kinase inhibitor p21, but not p27 or p57, is expressed in a subset of mononucleated cytotrophoblast cells (Genbacev et al., 2000). We detected by western blot that p21 expression, rather than that of p27, was elevated during BeWo cell fusion (Figure 4A). To determine whether p21 was necessary for cell fusion, p21 knockout (KO) BeWo cells were generated using a lentiviral CRISPR/Cas9 system (Figures S4A and S4B; Figure 4B). The fusion ability of p21-KO cells was dramatically lower (Figure 4C), and p21-KO cells showed lower expression of Syncytin-2 mRNA (Figure 4D) after forskolin treatment. To determine the mechanism by which p21 regulates Syncytin-2 transcription, we first tested the effect of p21 on the

Figure 3. Reduced Function of Syncytin-2-Induced Syncytia (A) Syncytin-2-induced syncytia were less resistant to Listeria monocytogenes invasion compared to forskolin (FSK)-induced syncytia in BeWo cells. Scale bar represents 10 mm. (B) Syncytin-2-induced cells produced less progesterone. Data are shown as means ± SD of three experiments (**p < 0.01). Syn-2, Syncytin-2. (C) Mitochondrial morphology. Fragmented mitochondria in FSK-induced, post-mitotic syncytia and elongated mitochondria in Syncytin-2-induced syncytia. White dotted line circled area represents syncytia. Scale bar represents 20 mm. (D) Western blot analysis using indicated antibodies. The respiratory chain component Cox IV serves as a mitochondrial loading control. (E) Localization of Syncytin-2 and Ki67 in human placental villi from a recurrent spontaneous abortion (RSA)-affected placenta and a gestation age-matched control by immunofluorescence. Arrowheads indicate cells that expressed Syncytin-2. Note the co-localization of Syncytin-2 and Ki67 in the RSA sample. Scale bar represents 50 mm. See also Figure S3.

activity of a Syncytin-2 promoter-luciferase reporter (Liang et al., 2010). Co-transfection of p21 could increase Syncytin-2 promoter activity (Figure 4E). Immunofluorescence further indicated that p21-positive cells exhibited higher levels of Syncytin-2 50 -long terminal repeat (LTR)-driven luciferase expression (Figure 4F). Notably, however, overexpression of p21 alone was

not sufficient to induce fusion (Figure S4C), although it did induce cell-cycle arrest (Figure S4D). Syncytin-2 is transcriptionally regulated by the glial cells missing homolog 1 (GCM1) transcription factor, the master transcription factor that is critical for the formation of syncytiotrophoblast (Liang et al., 2010). We therefore explored the potential

Cell Reports 21, 1150–1159, October 31, 2017 1155

Figure 4. Cell-Cycle Inhibitor p21 Regulates Syncytin-2 Expression (A) Western blot of BeWo cells treated with or without forskolin (FSK). (B) Western blot of control or p21 knockout BeWo cells. CTL, control cells that were transduced with lacZ-targeting sgRNA along with a CRISPR/Cas9 lentivirus. p21-KO, p21-knockout BeWo cells that were generated using the CRISPR/Cas9 lentivirus. (C) Cell-cell fusion index was calculated for forskolin-treated indicated BeWo cells, using E-cadherin immunostaining. White dotted lines mark the syncytia (left panel). Red, E-cadherin; blue, DAPI. Scale bar represents 50 mm. Data are shown as means ± SD of 3 experiments (**p < 0.01). (D) p21-KO BeWo cells showed significantly lower expression of Syncytin-2 mRNA after forskolin treatment by real-time PCR. Data are shown as means ± SD of three experiments (**p < 0.01). (E) Luciferase assay of Syncytin-2 promoter activity. Data are shown as means ± SD of three experiments (**p < 0.01).

(legend continued on next page)

1156 Cell Reports 21, 1150–1159, October 31, 2017

synergistic effect of p21 and GCM1 in promoting Syncytin-2 gene transcription. Co-immunoprecipitation analyses showed that p21 interacted with GCM1 (Figure 4G). A FluoPPI (fluorescent-based technology detecting protein-protein interactions) assay, in which PPIs are visualized as foci, further confirmed the physical interaction between p21 and GCM1 (Figure 4H). Notably, live-cell imaging revealed that cells showing p21GCM1 interaction were able to fuse (Figure S4E). We next found that p21 and GCM1 synergistically enhanced Syncytin-2 promoter activity (Figure 4I), as shown by univariate ANOVA (p < 0.01). As a control, we added a nuclear export sequence (NES) to GCM1 that forced the exogenous GCM1 out of the nuclei (Figure 4J). The synergistic effects of p21 and GCM1 on Syncytin-2 transcription were blunted by GCM1-NES (Figure 4I). To test whether GCM1-p21 bound directly to the Syncytin-2 promoter, we used BeWo cells overexpressing Flag-tagged p21 to perform chromatin immunoprecipitation (ChIP) and found the binding of p21 to the Syncytin-2 promoter in the same region where GCM1 was reported to bind (Figure 4K). To explore the interactions between GCM1 and p21 in vivo, we performed immunostaining of first-trimester placental villi and found that p21-positive cells were generally Ki67-negative (Figure S4F) and p21 co-localized with GCM1 in the mononucleated cytotrophoblast cells (Figure 4L, left panel). Correspondingly, p21 expression was also detected within the Syncytin-2-expressing cells (Figures 4L, right panel, and 4M). Notably, while p21 was only detected in GCM1-positive cytotrophoblast cells, GCM1 was expressed in more cells, suggesting that expression of GCM1 precedes p21. DISCUSSION Although it is generally accepted that most syncytial cells are post-mitotic, how and why the syncytium maintains its postmitotic status remain unclear. Here, we demonstrate that cell-cycle exit happens before fusion and highlight the importance of the cell-cycle-restricted expression of the fusogenic protein Syncytin-2 for the structural and functional maintenance of human post-mitotic syncytiotrophoblast cells. The fact that G0 arrest is required for Syncytin-2 induction implies that a portion of G0 cells express Syncytin-2 and thus are able to fuse, whereas

proliferating cells do not express fusogenic proteins and are fusion-defective. Notably, forcing fusion of cells other than those in G0 results in unstable syncytia that exhibit compromised functions including the ability to resist pathogen invasion and secrete hormones such as progesterone. Restricted expression of Syncytin-2 in G0-arrested cells appears to be a mechanism to ensure maintenance of post-mitotic syncytia. It is important to note that using primary cells to do the in vitro experiments in this study was not an option because primary cytotrophoblast cells contain a very limited number of cycling cells (Kliman et al., 1986). The BeWo cell line may certainly have limitations as they are tumor derived and have been passaged for a long time. Having said that, it is important to note that BeWo cells retain the ability to exit the cell cycle on stimulus, to fuse and initiate the appropriate program of cell differentiation consistent with what syncytiotrophoblast cells do in vivo, including protection from pathogens and hormone production. Importantly, we used BeWo cells as a tool but validated our observations on human placental tissues by doing immunostaining. Other trophoblast cell lines like JEG3 cells or HTR8/SVneo cells generally do not fuse. Trophoblast stem cell (TSC) would be a good choice, but there are no established stable lines of ‘‘true’’ human TSCs (Gamage et al., 2016). There are reports on inducing human embryonic stem cells into trophoblast cells by BMP4 (Yabe et al., 2016), but the intermediates from embryonic stem cells (ESCs) to trophoblast cells, if they exist, are still unclear. Thus, BeWo cell would be the only appropriate choice currently for investigating the relationship between cell-cycle progression and cell-cell fusion, as long as conclusions are validated by staining of trophoblast cells in situ. Notably, the best tool to study human placental trophoblast fusion is still human TSC. Due to its unavailability now, efforts should be made to improve primary placental cultures. For instance, optimizing the culture condition to obtain and maintain more proliferative cytotrophoblast cells isolated from human placentas. It would be important to further study the relationship between cell-cycle progression and cellcell fusion in these systems, and further integrate our findings into reported pathways within the human placenta. The fact that human syncytiotrophoblast cells are Ki67 negative indicates that the syncytia is in the G0 phase. We provide evidence in this study to show that this is maintained by fusing

(F) Upper panel: schematic illustration of the vectors that express the mNG-p21 fusion protein and luciferase reporter for Syncytin-2 50 -LTR activity. Lower panel: immunofluorescence to detect the expression of luciferase reporters. Blue: the nuclear-localized mTagBFP2 indicating the transfected reporter construct. Red: immunofluorescence using an anti-luciferase antibody. Scale bar represents 20 mm. (G) Co-immunoprecipitation of p21 with GCM1. (H) FluoPPI assay indicating the interaction between p21 and GCM1. Ash-p21, Ash (homo-oligomerized protein assembly helper)-tagged p21; ZG1-GCM1, ZG1 (homo tetramer ZsGreen1)-tagged GCM1. Scale bar represents 15 mm. (I) Luciferase assay of Syncytin-2 promoter activity in transfected 293T cells. Data are shown as means ± SD of three experiments. a, b, c, d, different letters indicate statistical differences at p < 0.01, and the same letters indicate statistical differences at p > 0.05. (J) Immunostaining showing localization of p21 and GCM1 or GCM1-NES in BeWo cells transfected with Flag-tagged p21 and HA-tagged GCM1 or GCM1-NES. NES, nuclear export sequence. Scale bar represents 15 mm. (K) Association of p21 with Syncytin-2 promoter. BeWo-Flag-p21 (Tet-ON) cells were analyzed by ChIP assays in the absence of antibody (No Ab), presence of normal IgG, an anti-Flag antibody (Anti-Flag), or an anti-RNA polymerase II (Anti-RNA Pol II). The immunopurified DNA fragments were PCR amplified for promoter regions in the Syncytin-2 gene. As a control, a promoter region in the GAPDH gene was amplified. (L) Localization of p21, GCM1, and Syncytin-2 in the human placental villi from 7 weeks of gestation by immunofluorescence. Arrows indicate cells that express both p21 and GCM1, or both p21 and Syncytin-2, respectively. Scale bar represents 50 or 20 mm (with the white boxes). (M) Reconstructed z-stack confocal image representing higher resolution of the right panel of (L). Scale bar represents 20 mm. See also Figure S4.

Cell Reports 21, 1150–1159, October 31, 2017 1157

mononucleated cells also only in the G0 phase. It should be noted that, when there is abundant expression of fusion proteins, cells will fuse independently of the cell cycle (Podbilewicz et al., 2006). Therefore, the expression of fusogenic protein Syncytin-2 is confined within G0 phase to guarantee that cells in cell-cycle stages other than G0 phase will not fuse. It would be interesting to investigate whether the fusogenic protein restriction by the cell cycle applies to another family of fusogens—the F proteins in nematodes (Podbilewicz, 2014). However, due to the lack of detailed cell-cycle analysis in nematode cells prior to fusion, it is still unknown in which cell-cycle stages these cells fuse and whether there is also cell-cycle stage restriction in the expression of fusogenic proteins. Nevertheless, cell-cycle exit does seem to occur during other cell-cell fusion processes (Andre´s and Walsh, 1996; Euling and Ambros, 1996). The G0-fusogen restriction relies on, at least partially, the cellcycle inhibitor p21. Our results suggest that p21 may function as a selective Syncytin-2 coactivator via its interaction with GCM1, forming a complex that binds directly to the Syncytin-2 promoter and to enhance transcription. Whether p21 alone may also be able to activate the promoter activity of Syncytin-2 independent of GCM1 is unclear. In our transfection experiments in the nontrophoblast 293T cell line to assess Syncytin-2 promoter activity, p21 alone could increase transcription but p21 and GCM1 together had synergistic effects. Our observations highlight the ability of p21 to enhance fusogen induction while guaranteeing G0 arrest before fusion, but p21 alone is not sufficient to induce fusion. Notably, we have previously shown that GCM1-positive cells in mouse placenta were post-mitotic and GCM1 induced a rapid cell-cycle arrest when ectopically expressed in mouse TSCs (Cross et al., 2006). Given that p21-positive mononucleated cells are Ki67-negative in the human placenta, our data suggest that GCM1 may induce p21 expression to arrest the cell cycle, and in turn p21 enhances the function of GCM1 to begin the fusion program. When Syncytin-2 was inappropriately expressed in proliferating cells, we found that these cells fused to form syncytia that retained mitotic features. Of note, these syncytia were structurally and functionally compromised, thereby highlighting the importance of maintaining the post-mitotic state by G0restricted fusogen expression. Interestingly, it has been demonstrated that mitochondrial shape varies at different cell-cycle stages. Mitochondria in G0-arrested cells are more fragmented, while those in the G1-S transition are more elongated (Mitra et al., 2009). We found that the Syncytin-2-induced syncytia had abnormal mitochondria, and this likely contributed to their reduced ability to produce progesterone. The fact that the expression of Syncytin-2 in proliferative cells was detected in the placentas of patients that had experienced recurrent spontaneous abortion suggests that fusion of trophoblast cells in the wrong stage of the cell cycle may lead to pregnancy abnormities. Whether the dysfunction is due to abnormal endocrine function or the inability of syncytiotrophoblast to structurally act as one unit is unclear. In addition to the importance for syncytiotrophoblast function, the post-mitotic state for multinucleated syncytiotrophoblast may also contribute to limiting placental expansion. The syncytiotrophoblast sheds aggregates of nuclei into the maternal circulation during pregnancy, and it has been proposed

1158 Cell Reports 21, 1150–1159, October 31, 2017

that, if the syncytiotrophoblast was still proliferative, the mother may suffer from excessive invasion and chimerism (Benirschke et al., 2012). Collectively, our findings indicate that fusogenic proteins enable cells to fuse, but they alone will not guarantee the appropriate function of the formed syncytia. Fusogenic protein expression must be restricted to the G0 phase in order to ensure proper function of the fused cells. This study begins to uncover the mechanisms that interconnect the cell cycle, fusogen regulation, fusion, and functional regulation of trophoblast cells, and provides insights into other cell-cell fusion events. EXPERIMENTAL PROCEDURES Cell Fusion Assay and Related Analyses BeWo, BeWo-FucciNG, or p21-KO BeWo cells were treated with 25 mM forskolin (Sigma-Aldrich) for 48 or 72 hours to trigger fusion, and cells were then subjected to real-time PCR, western blot, Listeria monocytogenes invasion assay, progesterone assay, immunofluorescence microscopy, or timelapse microscopy. See also Supplemental Experimental Procedures. Human Tissue Collection Normal human placental tissues (n = 5; 6–8 weeks of gestation) and miscarriage samples (n = 12, 7–12 weeks) diagnosed as recurrent spontaneous abortion along with their age-matched controls (n = 8) were collected in accordance with the policy of the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, and The Second Hospital of Tianjin Medical University. All of the normal placentas were obtained from female patients who underwent legal abortion. Informed consent was obtained from all women who donated their placentas. Samples were used according to standard experimental protocols that were approved by the Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. The placental villi were fixed and subjected to immunostaining and microscopy. See also Supplemental Experimental Procedures. Statistical Analysis Each experiment was repeated independently at least three times. Differences among groups were evaluated by one-way ANOVA or t-test analyses. To test potential synergistic effects of p21 and GCM1 on Syncytin-2 transcription, we performed univariate ANOVA. Analyses were conducted using the Statistical Package for Social Science (SPSS for Windows package release 16.0; SPSS). Data are presented as the mean ± SD. A p value of less than 0.05 was considered to be statistically significant. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and four movies and can be found with this article online at https://doi.org/10.1016/j.celrep.2017.10.019. AUTHOR CONTRIBUTIONS X.L., J.C.C., and H.W. conceptualized the project. X.L., R.W., and Y.G. designed and performed experiments. X.L., R.W., C.Z., H.W., H.-Y.L., Y.G., J.C.C., and H.W. performed data analysis. X.L., J.C.C., and H.W. wrote the original draft. X.L., R.W., H.-Y.L., J.C.C., and H.W. reviewed and edited the manuscript. ACKNOWLEDGMENTS We thank Drs. Nathan Shaner, Hiroyuki Miyoshi, Feng Zhang, Tao Xu, Guangshuo Ou, Hungwen Chen, Vladislav Verkhusha, and Tiebang Kang for providing vectors and cDNAs. We thank Shiwen Li for technical assistance in confocal photography. We thank Hua Qin and Tong Zhao for flow cytometry experiments. This research was supported by grants from the National Natural

Science Foundation of China (81490741) and the National Key Research and Development Program of China (2017YFC1001401 and 2016YFC1000208).

Liang, C.Y., Wang, L.J., Chen, C.P., Chen, L.F., Chen, Y.H., and Chen, H. (2010). GCM1 regulation of the expression of syncytin 2 and its cognate receptor MFSD2A in human placenta. Biol. Reprod. 83, 387–395.

Received: August 9, 2016 Revised: September 12, 2017 Accepted: October 4, 2017 Published: October 31, 2017

Mitra, K., Wunder, C., Roysam, B., Lin, G., and Lippincott-Schwartz, J. (2009). A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase. Proc. Natl. Acad. Sci. USA 106, 11960–11965.

REFERENCES Abe, T., Sakaue-Sawano, A., Kiyonari, H., Shioi, G., Inoue, K., Horiuchi, T., Nakao, K., Miyawaki, A., Aizawa, S., and Fujimori, T. (2013). Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter. Development 140, 237–246. Aguilar, P.S., Baylies, M.K., Fleissner, A., Helming, L., Inoue, N., Podbilewicz, B., Wang, H., and Wong, M. (2013). Genetic basis of cell-cell fusion mechanisms. Trends Genet. 29, 427–437. Andre´s, V., and Walsh, K. (1996). Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J. Cell Biol. 132, 657–666. Benirschke, K., Burton, G.J., and Baergen, R.N. (2012). Basic structure of the villous trees. In Pathology of the Human Placenta (Springer), pp. 55–100. Blaise, S., de Parseval, N., Be´nit, L., and Heidmann, T. (2003). Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution. Proc. Natl. Acad. Sci. USA 100, 13013–13018. Burton, G.J., and Jauniaux, E. (1995). Sonographic, stereological and Doppler flow velocimetric assessments of placental maturity. Br. J. Obstet. Gynaecol. 102, 818–825. Cross, J.C., Werb, Z., and Fisher, S.J. (1994). Implantation and the placenta: key pieces of the development puzzle. Science 266, 1508–1518. Cross, J.C., Nakano, H., Natale, D.R., Simmons, D.G., and Watson, E.D. (2006). Branching morphogenesis during development of placental villi. Differentiation 74, 393–401. Esnault, C., Cornelis, G., Heidmann, O., and Heidmann, T. (2013). Differential evolutionary fate of an ancestral primate endogenous retrovirus envelope gene, the EnvV syncytin, captured for a function in placentation. PLoS Genet. 9, e1003400. Euling, S., and Ambros, V. (1996). Heterochronic genes control cell cycle progress and developmental competence of C. elegans vulva precursor cells. Cell 84, 667–676. Gamage, T.K., Chamley, L.W., and James, J.L. (2016). Stem cell insights into human trophoblast lineage differentiation. Hum. Reprod. Update 23, 77–103. Genbacev, O., McMaster, M.T., and Fisher, S.J. (2000). A repertoire of cell cycle regulators whose expression is coordinated with human cytotrophoblast differentiation. Am. J. Pathol. 157, 1337–1351.

Podbilewicz, B. (2014). Virus and cell fusion mechanisms. Annu. Rev. Cell Dev. Biol. 30, 111–139. Podbilewicz, B., Leikina, E., Sapir, A., Valansi, C., Suissa, M., Shemer, G., and Chernomordik, L.V. (2006). The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo. Dev. Cell 11, 471–481. Richart, R. (1961). Studies of placental morphogenesis. I. Radioautographic studies of human placenta utilizing tritiated thymidine. Proc. Soc. Exp. Biol. Med. 106, 829–831. Robbins, J.R., Skrzypczynska, K.M., Zeldovich, V.B., Kapidzic, M., and Bakardjiev, A.I. (2010). Placental syncytiotrophoblast constitutes a major barrier to vertical transmission of Listeria monocytogenes. PLoS Pathog. 6, e1000732. Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., et al. (2008). Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498. Schneider, J.W., Gu, W., Zhu, L., Mahdavi, V., and Nadal-Ginard, B. (1994). Reversal of terminal differentiation mediated by p107 in Rb/ muscle cells. Science 264, 1467–1471. Shaner, N.C., Lambert, G.G., Chammas, A., Ni, Y., Cranfill, P.J., Baird, M.A., Sell, B.R., Allen, J.R., Day, R.N., Israelsson, M., et al. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409. Shcherbakova, D.M., and Verkhusha, V.V. (2013). Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754. Simpson, R.A., Mayhew, T.M., and Barnes, P.R. (1992). From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: a study of nuclear number using the disector. Placenta 13, 501–512. Wang, R., Dang, Y.L., Zheng, R., Li, Y., Li, W., Lu, X., Wang, L.J., Zhu, C., Lin, H.Y., and Wang, H. (2014). Live cell imaging of in vitro human trophoblast syncytialization. Biol. Reprod. 90, 117. Wasilewski, M., Semenzato, M., Rafelski, S.M., Robbins, J., Bakardjiev, A.I., and Scorrano, L. (2012). Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts. Curr. Biol. 22, 1228–1234.

Hahlin, M., Wallin, A., Sjo¨blom, P., and Lindblom, B. (1990). Single progesterone assay for early recognition of abnormal pregnancy. Hum. Reprod. 5, 622–626.

Yabe, S., Alexenko, A.P., Amita, M., Yang, Y., Schust, D.J., Sadovsky, Y., Ezashi, T., and Roberts, R.M. (2016). Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas. Proc. Natl. Acad. Sci. USA 113, E2598–E2607.

Kliman, H.J., Nestler, J.E., Sermasi, E., Sanger, J.M., and Strauss, J.F., 3rd. (1986). Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118, 1567–1582.

Yerushalmi, R., Woods, R., Ravdin, P.M., Hayes, M.M., and Gelmon, K.A. (2010). Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 11, 174–183.

Cell Reports 21, 1150–1159, October 31, 2017 1159