
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38483994
202318176
10.1073/pnas.2318176121
research-articleResearch Articledev-bioDevelopmental Biology412
Biological Sciences
Developmental Biology
Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development
Kong Xuhui a b 1 https://orcid.org/0000-0002-2042-4535

Li Ruiqi c d e 1 https://orcid.org/0000-0003-0196-1965

Chen Manqi a b 1
Zheng Rongyan a b
Wang Jichang a b https://orcid.org/0000-0002-7027-0298

Sun Chuanbo bobo_1110@163.com
f 2
Qu Yuliang quyliang@mail.sysu.edu.cn
a b 2 https://orcid.org/0000-0002-6203-0032

aAdvanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
bKey Laboratory for Stem Cells and Tissue Engineering, Sun Yat-sen University, Ministry of Education, Guangzhou 510080, China
cReproductive and Genetic Hospital of Kapok, Hainan 571400, China
dDepartment of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
eThe First People’s Hospital of Kashgar, Kashgar 844000, China
fLaboratory of Medical Systems Biology, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, China
2To whom correspondence may be addressed. Email: bobo_1110@163.com or quyliang@mail.sysu.edu.cn.
Edited by Thomas Spencer, University of Missouri, Columbia, MO; received October 19, 2023; accepted February 12, 2024

1X.K., R.L., and M.C. contributed equally to this work.

14 3 2024
19 3 2024
14 9 2024
121 12 e231817612119 10 2023
12 2 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

HERVH represents an engaging member of co-opted retroviral elements in our genome. Despite its prominent roles in cell pluripotency and epiblast development, whether and how HERVH contributes to other developmental events remains rarely explored. Here, we revealed that the LTR7C subfamily of HERVH (LTR7C/HERVH) is highly transcribed and plays a crucial role in human trophoblast development. Remarkably, ectopic expression of Urothelial Cancer Associated 1, a primate-specific long non-coding RNA (lncRNA) derived from the LTR7C/HERVH, impedes human trophoblast syncytialization and is detectable in EO-PE placenta, suggesting a pathological role of dysregulated HERVH. Our study has broadened the function of HERVH in early human development and unveiled a unique mechanism by which ERVs shape human placentation beyond acting as regulatory cis-elements.

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.

endogenous retrovirus
Urothelial Cancer Associated 1
trophoblast stem cell
trophoblast syncytialization
preeclampsia
==== Body
pmcThe human placenta is an extraembryonic organ that not only supports embryo development but also exerts a durable influence on the fitness of both the mother and fetus (1–3). The human placenta develops from the trophectoderm (TE) and contains three major trophoblast subpopulations: villus cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs) (4). CTBs give rise to multinucleated STBs through the cell–cell fusion procedure (also termed trophoblast syncytialization). Localized at the outer layer of the villus, the STBs contribute to substance exchange between the fetus and mother and produce plentiful steroid and polypeptide hormones essential for embryonic development and pregnancy. In addition, the distal CTB cluster in the cell column of anchoring villi can differentiate into EVTs, which properly invade the maternal decidua and remodel maternal spiral arteries for sufficient nutrient supply. Abnormal STB formation and/or EVT invasion has been implicated in multiple placental dysfunction syndromes such as early-onset preeclampsia (EO-PE) (5, 6), fetal growth restriction (FGR) (7, 8), and miscarriage (9, 10). Thus, a better understanding of human placental development would provide insights into overcoming these pregnancy complications.

Notwithstanding their functional similarities, placental organs are highly divergent in their morphological and molecular features across mammalian species (11, 12). Emerging evidence suggests that species-specific endogenous retroviruses (ERVs), a subclass of retrotransposable elements originating from ancient retroviral infection, are a driving force for placental evolutionary diversification (13–15). Human ERVs (HERVs) constitute approximately 8% of the modern genome and are mainly silenced by the genomic surveillance machinery. Intriguingly, ERVs have been recognized to be frequently derepressed and contribute to a series of mammalian developmental events including zygotic genome activation (ZGA), lineage separation, and placentation (15–18). One notable example is the ERV envelope gene (env)-coded proteins (e.g., syncytin and suppressyn) which actively promote trophoblast syncytialization, proliferation, and antiviral response (19–22). In addition to protein-coding potential, the prominent cis-regulatory activity of ERVs as species-specific enhancers has been well appreciated in rodent and human trophoblast stem cells (TSCs) (13, 14, 23–26). However, little is known about whether ERVs control human trophoblast development through other mechanisms.

Primate-specific HERVH promotes the proliferation and pluripotency of human epiblast and pluripotent stem cells (hPSCs) (27–31). HERVH consists of several subfamilies (LTR7, 7B, 7C, 7Y, and others), displaying stage-specific expression patterns over human preimplantation development (32, 33). HERVH not only embeds motifs bound by pluripotent factors but also provides topologically associating domain (TAD) boundaries and chimeric transcripts to maintain pluripotency (34–36). More recently, HERVH transcription has been linked to genome stability of human inner cell mass (ICM) (31). Nevertheless, whether and how HERVH contributes to the development of other lineages remains elusive.

In this study, we systematically examined the expression pattern of HERVH in early human development and found that the LTR7C/HERVH subfamily is transcriptionally active in the trophoblast lineage. Particularly, Urothelial Cancer Associated 1 (UCA1), a long non-coding RNA (lncRNA) derived from LTR7C/HERVH subfamily, is highly expressed in human trophoblasts. UCA1 plays a crucial role in hTSC self-renewal and EVT differentiation, whereas its ectopic expression induces the interferon signaling pathway and impedes trophoblast syncytialization. Importantly, the transcriptome of human STBs with UCA1 overexpression shares substantial characteristics with that of EO-PE placenta, supporting hyperactive UCA1 as a promising biomarker of this disease.

Results

LTR7C/HERVH Exhibits Regulatory and Transcriptional Activity in Human Trophoblasts.

Previous studies have revealed that distinct HERVH subfamily could be divergently expressed in human preimplantation embryos and hPSCs (27, 32, 33). Here, we interrogated the transcriptional potential of HERVH in extraembryonic lineages, particularly in trophoblasts. To this end, we first examined the chromatin accessibility of HERVH-related LTRs by performing ATAC-seq of human trophoblast stem cells (hTSCs), combined with data mining on the online-available resources (37). As expected, consistent with their transcriptional activity (32, 33), the LTR7, LTR7B, and LTR7Y loci displayed an open chromatin state in human ICM (Fig. 1A). On the contrary, LTR7C exhibited relatively accessible chromatin in hTSCs (Fig. 1A), corresponding to a recent report showing that LTR7C is enriched for active transcription associated H3K27ac in hTSCs (SI Appendix, Fig. S1A) (26). Besides, by mining the CUT&Tag data of trophoblast transcription factors (26), we found that the binding motifs of TEAD4, TFAP2C, and GATA3 were more enriched for LTR7C sequences (Fig. 1B), suggesting a potentially cis-regulatory function of LTR7C in hTSCs. Furthermore, using the Genomic Regions Enrichment of Annotations Tool (GREAT), we identified candidate nearby genes potentially regulated by LTR7C and found that those expressed genes were significantly enriched for N-glycan biosynthesis and viral life cycle pathways (Fig. 1C and SI Appendix, Fig. S1B). On the other hand, the single-cell RNA-seq analysis of human early embryo (38, 39) revealed that the LTR7C transcription was gradually increased in postimplantation trophoblast subtypes, including CTB, EVT, and STB as well as hTSCs (SI Appendix, Fig. S1 C and D). Altogether, these data indicated that the LTR7C/HERVH elements not only exhibit regulatory potential but also are actively transcribed in human trophoblast lineage.

Fig. 1. LTR7C/HERVH-derived UCA1 is transcribed in human trophoblasts. (A) Chromatin accessibility signal of HERVH subfamilies in the human inner cell mass (hICM), human embryonic stem cell (hESC), human trophectoderm (hTE), and human trophoblast stem cell (hTSC), the blue color density represents Reads Per Kilobase of transcript per Million mapped reads (RPKM). (B) Dot plot showing the ratio of trophoblast transcription factor-bound loci on LTRs of HERVH subfamilies. (C) Top ten enriched KEGG pathways of the candidate genes regulated by LTR7C in hTSC. (D) Heatmap showing normalized expression level of HERVH LTRs-derived lncRNAs (corresponding to the cluster 6 and 7 in SI Appendix, Fig. S1E) in early human embryo. (E) Genome browser snapshot showing chromatin landscape and mRNA expression of LTR7C/HERVH-derived UCA1 in hTSC.

LTR7C/HERVH-Derived UCA1 Is Essential for hTSC Proliferation.

Next, to investigate whether HERVH exerts a function in human trophoblasts beyond acting as cis-regulatory elements, we focused on the HERVH-derived long non-coding RNAs (lncRNAs). To this end, we examined the expression of distinct HERVH subfamily-derived lncRNAs in early human embryos and identified two clusters (cluster 6 and 7) that exhibited relatively high expression in trophoblast lineage (SI Appendix, Fig. S1E). Notably, the LTR7C/HERVH-derived UCA1 ranked the most highly expressed lncRNA in postimplantation trophoblasts (Fig. 1D). Consistently, with accessible chromatin state and strong binding signals of trophoblast transcription factors (e.g., GATA3, TEAD4, and TFAP2C), UCA1 was actively transcribed in hTSCs and early trophoblasts (Fig. 1E and SI Appendix, Fig. S1 H and I), whereas ESRG, a LTR7/HERVH-derived lncRNA (27), was specially expressed in pluripotent epiblast (SI Appendix, Fig. S1 G and J), confirming the divergent expression of HERVH subfamilies.

UCA1 has been widely implicated in carcinogenesis (40, 41), but little is known about its role in human trophoblast development. With trophoblastic tumor or immortal cell-based models, previous studies have suggested that UCA1 might be involved in multiple trophoblast phenotypes such as proliferation, apoptosis, cell migration, and cell fusion (42–45). In this study, we systematically investigated the biological function of UCA1 using the well-established hTSC model (46), which can recapitulate the characteristics of postimplantation CTBs (47). First, the cell colony formation showed that the knockdown (KD) of UCA1 led to a decreased hTSC proliferation rate (Fig. 2 A and B). However, the RT-qPCR and immunostaining data showed no obvious change in the expression levels of hTSC signature genes (e.g., GATA2, GATA3, DLX3, KRT18, TEAD4, ITGA6, and ELF5) (Fig. 2 C and D), indicating that UCA1 depletion has little influence on hTSC cellular identity. Transcriptomic profiling revealed that multiple signaling pathways were up-regulated upon UCA1 KD, including cell adhesion molecules, Ras signaling, and MAPK signaling, while the pathways responding to viral infection were suppressed such as those related to herpes simplex virus 1 infection, hepatitis C, and influenza A (Fig. 2E), suggesting that UCA1 might play a role on modulating immune response in hTSCs. On the other hand, we examined the differentiation potential of hTSCs upon UCA1 KD and found that the expression of EVT signature genes such as FN1, MMP2, and HLA-G was slightly increased (SI Appendix, Fig. S2 A and B), whereas UCA1 depletion had marginal effect on expression of STB signature genes in either 2D or 3D culture conditions (SI Appendix, Fig. S2 C–E). To further evaluate the trophoblast syncytialization, we co-cultured mCherry- and GFP-labeled hTSCs and quantified the fused cells with double-positive signals during STB differentiation (SI Appendix, Fig. S2F). The quantification data showed that the fusion ratio of co-cultured cells in STB medium was much higher than that from co-culture in hTSC medium (SI Appendix, Fig. S2 G–I), indicating an efficient STB induction. However, consistent with the RT-qPCR data (SI Appendix, Fig. S2 C and D), there was no significant difference in STB fusion between control and UCA1 KD co-culture (SI Appendix, Fig. S2 G–I). Altogether, the loss-of-function analysis suggested that UCA1 is essential for hTSC proliferation.

Fig. 2. UCA1 is essential for hTSC proliferation. (A) Colony formation assay showing hTSC proliferation upon UCA1 KD. (B) Quantification of cell colonies formed in (A). Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using unpaired t test, *P < 0.05, **P < 0.01. (C) RT-qPCR analysis showing mRNA expression of hTSC-related genes upon UCA1 KD. Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using an unpaired t test, ****P < 0.0001. (D) Representative immunostaining of GATA3, GATA2, and ITGA6 in hTSCs upon UCA1 KD. (Scale bar, 100 μm.) (E) Bubble plot showing KEGG pathways enriched upon UCA1 KD. P values were determined by the Kolmogorov–Smirnov test. Bubble size represents the number of enriched genes, and the color represents statistical significance.

Human Trophoblast Syncytialization Is Blocked upon UCA1 Overexpression.

To gain more insight into the biological role of UCA1 in trophoblasts, we further performed gain-of-function analysis of UCA1 in hTSCs. As opposed to the phenotype of UCA1 KD (Fig. 2 A and B), UCA1 overexpression could promote hTSC proliferation (SI Appendix, Fig. S3 A and B), confirming the potential role of UCA1 on hTSC self-renewal. On the other hand, the RT-qPCR and immunofluorescence analyses suggested that UCA1 overexpression had marginal effect on hTSC identity and EVT differentiation (SI Appendix, Fig. S3 C and D). In contrast, despite the large syncytium-like morphology, the STBs differentiated from UCA1-overexpressing hTSCs maintained mononucleated, suggesting a deficiency in STB formation (Fig. 3A). Indeed, the STB signature genes (e.g., SDC1, INHA, CGA, CGB, ERVV-1, and ERVW-1) were comprehensively down-regulated upon UCA1 ectopic expression (Fig. 3B). In addition, the immunofluorescence analysis showed that the STB fusion was impaired upon UCA1 overexpression (Fig. 3 C and D). The fusion quantification assay further revealed that compared with the control population, the percentage of double-positive cells dramatically declined in the UCA1-overexpressing STB group (30.7% to 17.7%) (Fig. 3 E and F), suggesting that trophoblast fusion was blocked following UCA1 overexpression. The ERV envelope gene-coded syncytin protein is known to promote cell fusion of trophoblasts and 293 T cells (48, 49). Ectopic UCA1 was able to decrease the syncytin-1 (ERVW-1)-mediated 293T cell fusion (74.3% to 38.9%) (SI Appendix, Fig. S3E), indicating that UCA1 overexpression could negatively regulate the syncytin-mediated STB formation.

Fig. 3. Ectopic UCA1 despairs STB formation. (A) Representative bright-field images showing the cellular morphology of STBs overexpressing UCA1. The white arrow labels the mononucleated syncytium. (Scale bar, 100 μm.) (B) RT-qPCR analysis showing mRNA expression of STB-related genes upon UCA1 overexpression. Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using an unpaired t test, *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Representative immunostaining showing STB formation. The white arrow labels the multinucleated STB (Scale bar, 100 μm.) (D) Quantification of the STB formed in (C). Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using an unpaired t test, *P < 0.05. (E) Schematic diagram demonstrating the strategy to quantify STB formation by FACS. See experimental details in Methods. (F) Representative FACS plot from three biological replicates (n = 3) showing the percentage of induced STBs upon UCA1 overexpression.

UCA1 Overexpression Impairs Trophoblast Syncytialization Coinciding with the Increased Interferon Signaling.

To investigate the mechanism whereby UCA1 overexpression inhibits STB formation, we performed the bulk transcriptome profiling of resultant STBs. Notably, multiple genes related to interferon (IFN) signaling were significantly up-regulated upon UCA1 overexpression (Fig. 4 A-B), including those responsible for IFNα/β production, such as myeloid differentiation primary response protein 88 (MYD88), and those acting downstream of type I IFN signaling, such as type I IFN receptor subunits 1/2 (IFNAR1/2), Janus kinase 1 (JAK1), signal transducer and activator of transcription 1/2/3 (STAT1/2/3), IFN-stimulated gene 15 (ISG15), and IFN-induced transmembrane protein 3 (IFITM3)( SI Appendix, Fig. S4A). Intriguingly, ectopic UCA1-induced IFN signaling seemed more robust in STBs compared to that in hTSCs or the HTR-8 trophoblast cell line (Fig. 4B and SI Appendix, Fig. S4B). In line with this, compared to the transcriptomes of primary EVTs or CTBs, the overrepresented genes in STBs were enriched for the type I interferon signaling pathway or viral transcription, respectively (SI Appendix, Fig. S4 C-D). Besides, the gene ontology (GO) analysis revealed that upon UCA1 overexpression, the up-regulated genes were enriched for pathways related to oxidative phosphorylation, ATP synthesis, type I interferon production, and NF-kB signaling (Fig. 4C). Correspondingly, the gene set enrichment analysis (GSEA) also confirmed that the type I interferon signaling was increased during STB induction from hTSCs overexpressing UCA1 (Fig. 4D).

Fig. 4. Ectopic UCA1 induces IFN-induced immune signaling and blocks STB formation. (A) Heatmap showing mRNA expression of IFN-related genes upon UCA1 overexpression. (B) RT-qPCR analysis showing the mRNA expression of IFN-related genes in the hTSCs and STBs. Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using an unpaired t test, *P < 0.05, **P < 0.01. (C) Bar plot showing GO items of up-regulated genes upon UCA1 overexpression in STBs. (D) GSEA plot showing that type I interferon signaling was increased upon UCA1 overexpression in STBs. NES, normalized enrichment score. Adjusted P value = 0.036. (E) Representative western blot showing the shRNA-mediated decrease of IFITM3 in the STBs overexpressing UCA1. (F) Representative immunofluorescence showing STB formation. The dashed circle labels fused STBs. (Scale bar, 100 μm.) (G) Representative FACS plot from three biological replicates (n = 3) showing the percentage of STB formation. (H) ELISA measurement of hCG in the culture medium of STBs. Data are mean ± SEM from three biological replicates (n = 3). P values were calculated using one-way ANOVA followed by Tukey’s multiple tests, *P < 0.05. ns, not significant.

IFN signaling is well known for its ability to restrict virus infection across a broad range of cell types, including placental trophoblasts (50, 51). Importantly, IFN-induced IFITMs (i.e., IFITM1/2/3) can compromise the syncytin-mediated STB formation and trophoblast invasion, likely contributing to abnormal placental development caused by increased type I interferon signaling in multiple pregnancy complications (48, 49, 51, 52). Given the prominent role of IFITMs in controlling STB formation, we hypothesized that ectopic UCA1 could block trophoblast syncytialization by increasing the expression of IFITMs. To test this hypothesis, we examined whether the defective syncytialization could be rescued by silencing IFITM3 during STB differentiation from hTSC overexpressing UCA1 (Fig. 4E). The IFITM3 decrease had no influence on UCA1 expression, confirming that IFITM3 could function downstream of UCA1 (SI Appendix, Fig. S4E). Notably, the quantification data showed that the cell fusion of hTSCs overexpressing UCA1 was significantly improved following IFITM3 silencing (Fig. 4 F and G). In addition, the hCG concentration could be rescued to levels comparable to those of the control (Fig. 4H). Collectively, these data suggested that ectopic UCA1 might inhibit trophoblast syncytialization, at least in part, through eliciting the “IFN-IFITM3” signaling pathway.

UCA1 Upregulation Is Implicated in EO-PE Pathogenesis.

Considering the dysregulation of ERVs in placenta-related pregnancy complications (53, 54), we further interrogated the pathological role of ERVs in EO-PE. To this end, we reanalyzed the online available placental transcriptomes from EO-PE patients (55) and identified multiple dysregulated ERV elements in EO-PE placenta such as LTR7C, HERVK3, Harlequin, and LTR3A (Fig. 5 A-B). Correspondingly, the LTR7C/HERVH-derived UCA1 was significantly up-regulated in EO-PE placenta, which is consistent with previous studies (42, 56) (Fig. 5C). Recent evidence has suggested that abnormal trophoblast syncytialization is involved in EO-PE development (57–59). Indeed, comparative transcriptome profiling showed that the up-regulated genes in EO-PE seemed to be more enriched for the STB overrepresented genes, including UCA1 (Fig. 5D). Of note, the protein interaction prediction of those EO-PE-enriched STB genes identified a strong interactome network related to hormone production (SI Appendix, Fig. S5A), including LEP, CRH, ENG, ANG, CGB5, and CGB8. Consistently, the GO analysis indicated that the up-regulated genes in EO-PE placenta were enriched for vesicle organization, viral process, NF-kB signaling, and type I interferon-mediated signaling (Fig. 5E), supporting the potential role of hyperactive type I interferon signaling in EO-PE pathogenesis.

Fig. 5. UCA1 upregulation is implicated in EO-PE. (A) Volcano plot showing differentially expressed LTRs in EO-PE placenta. The marked points represent the highest or lowest magnitudes within EO-PE placenta. (B) Representative dysregulated LTRs in EO-PE placenta. (C) UCA1 is up-regulated in EO-PE placenta. RPKM, Reads Per Kilobase of transcript per Million. ****P < 0.0001. (D) Comparative transcriptome analysis of CTB, STB, EVT, and EO-PE placenta. UCA1 expression is relatively increased in STB and EO-PE placenta. (E) Bar plot showing representative GO items of up-regulated genes in EO-PE placenta. (F) The magenta cluster exhibiting a positive correlation with the occurrence of EO-PE (the correlation was 0.72 and P =2e-07). (G) Cluster analysis between EO-PE placenta and STB overexpressing UCA1 based on transcriptome similarity. (H) Co-dysregulated genes between EO-PE placenta and the STBs overexpressing UCA1.

Given the inhibitoryrole of UCA1 upregulation in STB formation, we hypothesized that STBs overexpressing UCA1 could recapitulate some features of EO-PE placenta. To test this hypothesis, we first performed the weighted gene co-expression network analysis (WGCNA) and identified one gene cluster showing a positive relationship to the EO-PE placental transcriptome (Fig. 5F and SI Appendix, Fig. S5B), including UCA1, DLX6, MMP9, FLT1, ENG, GATA3, INHBA, and INHA. Furthermore, the cluster analysis revealed that the EO-PE placental samples were prone to cluster together with the STBs overexpressing UCA1 (Fig. 5G), suggesting certain molecular similarities between the two samples. Consistently, the co-up-regulated genes between the two samples included RNF223, NLRP7, UCA1 and LY6D, while CXCL14 and AOC3 were co-down-regulated (Fig. 5H). The GO analysis further showed that the co-up-regulated genes were enriched for the positive regulation of cell–substrate adhesion, whereas the co-down-regulated genes were enriched for cell cycle–related pathways (SI Appendix, Fig. S5 C and D). Altogether, these data suggested that the STBs overexpressing UCA1 exhibit some molecular features of EO-PE placenta.

Discussion

HERVH has long been recognized as a crucial element to maintain the pluripotency of human epiblast and hPSCs. Here, we identified an unappreciated role of HERVH in shaping human trophoblast development. Compared to other subfamilies, the LTR7C/HERVH subfamily displays accessible chromatin and is highly transcribed in human trophoblasts. It has been reported that distinct HERVH subfamily could be variably expressed in human preimplantation embryo and hPSCs (32, 33). The transcriptional discrepancy could be attributed to the extensive diversity of LTR sequences of HERVH, potentially caused by point mutations, indels, and recombination events (32). Sequence heterogeneity has been proposed to generate a unique combination of transcription factor binding motifs, leading to stage-specific expression of a defined HERVH subfamily in early human development (32). Consistently, several key trophoblast transcription factors, including TEAD4, TFAP2C, and GATA3, preferentially bind LTR7C elements in hTSCs. Thus, the binding to transcriptional activators could define the unique reactivation of the LTR7C/HERVH subfamily in the human trophoblast lineage. Another explanation for the differential expression pattern of the LTR7C subfamily is that those elements might escape the silencing effect exerted by transcriptional repressors such as the KZFPs-KAP1 complex, which can recognize ERVs in a sequence-specific manner (16, 32). It is tempting to speculate that the unappreciated vacancy of KZFPs might allow LTR7C/HERVH to be derepressed in human trophoblast lineage.

Emerging evidence has proposed that ERVs play a cis-regulatory role in trophoblast stem cell maintenance and differentiation, mostly acting as species- and placenta-specific enhancers (14, 23, 24, 26, 60). In contrast, to explore the biological function of LTR7C/HERVH in human trophoblasts, we focused on the subfamily transcribed lncRNA UCA1. Although previous studies examined the function of UCA1 in trophoblast proliferation, fusion, and invasion (42–45), those trophoblast cell models are either choriocarcinoma cells (i.e., BeWo) or immortalized cells (i.e., HTR-8/SVneo) and cannot fulfill the criteria of human first-trimester trophoblast (61). Using the well-established hTSC model, an in vitro counterpart of human CTB (46, 47), we reveled that the proper expression of UCA1 plays a crucial role in trophoblast proliferation, whereas ectopic UCA1 could block the syncytin-1-directed trophoblast syncytialization. It has been reported that the infection of RNA virus Zika could impair placentation and fetal development through eliciting type I IFN signaling in mice (62). Consistently, the treatment of type I IFN, but not type III IFN, can induce robust ISG expression and abnormal STB morphology in human villous explants (62), suggesting a causal role of the hyperactive type I IFN in defective STB formation. Here, we proposed that as an ERV-derived lncRNA, ectopic UCA1 might mimic the infection of exogenous RNA virus to compromise STB formation by effectively triggering the type I IFN signaling.

On the other hand, ERV-derived nucleic acids can be sensed to activate type I IFN-directed innate immunity in tumorigenesis (60, 63). In addition, corresponding to its function in cancer cell proliferation and invasion (40, 64, 65), UCA1 could promote hTSC proliferation and likely inhibit EVT differentiation, suggesting a potentially conserved function of UCA1 in between trophoblast development and tumorigenesis. Indeed, considerable similarities between trophoblasts and cancer cells have been widely documented such as invasion, migration, and immune evasion (66–68). Thus, the hTSCs expressing ectopic UCA1 might represent a useful system for comparative study between placentation and cancer biology.

In addition to LTR7C/HERVH, we identified a set of unique HERV elements that are dysregulated in the EO-PE placenta such as HERVK3, LTR3A, and LTR77. Of particular interest, LTR3A has been recently reported to bind key placental transcription factors including GATA3, TEAD4, and TFAP2C (26), and its function in human trophoblasts is worth further investigation. Although the pathogenesis of EO-PE has long been thought to be attributed to shadow trophoblast invasion, emerging evidence has identified improper STB formation in this process (57–59). Of note, Admati et al. recently observed that more mononucleated STBs appeared in EO-PE placental villi, indicating compromised STB fusion (59). In line with this, our comparative transcriptomic analysis revealed that the up-regulated genes in EO-PE placenta are enriched for STB-related genes, including UCA1. Furthermore, the STBs overexpressing UCA1 share certain molecular features with the EO-PE placenta, suggesting a pathological role of ectopic UCA1 in this disease.

Taken together, our study unveiled the regulatory activity of LTR7C/HERVH and identified this subfamily-derived lncRNA UCA1 as a critical player during trophoblast development. The hTSCs overexpressing UCA1 represent a promising in vitro model to study human placentation and placenta-related diseases.

Methods

Ethics Approval.

Ethics approval for the study was granted by the Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University (2020 Reproductive Ethics No. 15). All participating patients were provided informed consent in accordance with institutional and national guidelines.

Culture of Human Trophoblast Stem Cells (hTSCs).

hTSCs were generated following the published protocol (46). Briefly, human blastocysts were cultured in a four-well plate pretreated with collagen IV for at least 30 min using G2 Plus medium (Vitrolife, Cat# 10132). Subsequently, the culture was switched to the hTSC medium and maintained in an environment consisting of 6% CO2, 5% O2, and 89% N2 for 7 d. The medium was changed every other day until colony formation occurred. Approximately 1 wk later, the clones were dissociated into individual cells using TrypLE and then seeded onto a plate coated with collagen IV (Thermo Fisher Scientific, Cat#17104019) in hTSC medium. For details of experimental methods, see SI Appendix, SI Methods and Tables S1 and S2.

Supplementary Material

Appendix 01 (PDF)

The study was funded by Guangdong Basic and Applied Basic Research Foundation (2021A1515010308 to R.L.), National Natural Science Foundation of China (82360311 to R.L.)

Author contributions

J.W. and Y.Q. designed research; X.K., R.L., M.C., R.Z., and Y.Q. performed research; X.K., M.C., J.W., and Y.Q. analyzed data; and X.K., M.C., J.W., C.S., and Y.Q. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

Raw and processed sequencing data generated in this study have been submitted to the National Genomics Data Center (https://ngdc.cncb.ac.cn/) (69) under accession number PRJCA020593. Some study data available (The trophoblast stem cell line produced in this study are derived from human blastocysts. Based on the regulations of Ministry of Science and Technology of China, the raw sequencing data can be publicly accessed under the number PRJCA020593 on the National Genomics Data Center website (https://ngdc.cncb.ac.cn/) (69) upon request.

Supporting Information

This article is a PNAS Direct Submission.
==== Refs
1 A. Fraser, J. M. Catov, Placental syndromes and long-term risk of hypertension. J. Hum. Hypertens. 37 , 671–674 (2023).36702879
2 J. D. Aplin, J. E. Myers, K. Timms, M. Westwood, Tracking placental development in health and disease. Nat. Rev. Endocrinol. 16 , 479–494 (2020).32601352
3 G. J. Burton, E. Jauniaux, The human placenta: New perspectives on its formation and function during early pregnancy. Proc. Biol. Sci. 290 , 20230191 (2023).37072047
4 M. Y. Turco, A. Moffett, Development of the human placenta. Development 146 , dev163428 (2019).31776138
5 E. A. Phipps, R. Thadhani, T. Benzing, S. A. Karumanchi, Pre-eclampsia: Pathogenesis, novel diagnostics and therapies. Nat. Rev. Nephrol. 15 , 275–289 (2019).30792480
6 G. J. Burton, C. W. Redman, J. M. Roberts, A. Moffett, Pre-eclampsia: Pathophysiology and clinical implications. BMJ 366 , l2381 (2019), 10.1136/bmj.l2381.31307997
7 X. Shao , Placental trophoblast syncytialization potentiates macropinocytosis via mTOR signaling to adapt to reduced amino acid supply. Proc. Natl. Acad. Sci. U.S.A. 118 , e2017092118 (2021).33402432
8 G. J. Burton, E. Jauniaux, Pathophysiology of placental-derived fetal growth restriction. Am. J. Obstet. Gynecol. 218 , S745–S761 (2018).29422210
9 T. Laisk , The genetic architecture of sporadic and multiple consecutive miscarriage. Nat. Commun. 11 , 5980 (2020).33239672
10 E. Dimitriadis, E. Menkhorst, S. Saito, W. H. Kutteh, J. J. Brosens, Recurrent pregnancy loss. Nat. Rev. Dis. Primers 6 , 98 (2020).33303732
11 C. Lavialle , Paleovirology of “syncytins”, retroviral env genes exapted for a role in placentation. Philos. Trans. R. Soc. Lond. B, Biol. Sci. 368 , 20120507 (2013).23938756
12 M. Hemberger, C. W. Hanna, W. Dean, Mechanisms of early placental development in mouse and humans. Nat. Rev. Genet. 21 , 27–43 (2019), 10.1038/s41576-019-0169-4.31534202
13 M. A. Sun , Endogenous retroviruses drive lineage-specific regulatory evolution across primate and rodent placentae. Mol. Biol. Evol. 38 , 4992–5004 (2021), 10.1093/molbev/msab223.34320657
14 E. B. Chuong, M. A. Rumi, M. J. Soares, J. C. Baker, Endogenous retroviruses function as species-specific enhancer elements in the placenta. Nat. Genet. 45 , 325–329 (2013).23396136
15 A. D. Senft, T. S. Macfarlan, Transposable elements shape the evolution of mammalian development. Nat. Rev. Genet. 22 , 691–711 (2021), 10.1038/s41576-021-00385-1.34354263
16 J. Pontis , Hominoid-specific transposable elements and KZFPs facilitate human embryonic genome activation and control transcription in naive human ESCs. Cell Stem Cell 24 , 724–735.e725 (2019).31006620
17 H. Yu , Dynamic reprogramming of H3K9me3 at hominoid-specific retrotransposons during human preimplantation development. Cell Stem Cell 29 , 1031–1050.e1012 (2022).35803225
18 A. Sakashita , Transcription of MERVL retrotransposons is required for preimplantation embryo development. Nat. Genet 55 , 484–495 (2023).36864102
19 S. Mi , Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403 , 785–789 (2000).10693809
20 J. L. Blond , An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J. Virol. 74 , 3321–3329 (2000).10708449
21 S. Blaise, N. de Parseval, L. Bénit, T. Heidmann, Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution. Proc. Natl. Acad. Sci. U.S.A. 100 , 13013–13018 (2003).14557543
22 J. A. Frank , Evolution and antiviral activity of a human protein of retroviral origin. Science 378 , 422–428 (2022).36302021
23 C. D. Todd, Ö. Deniz, D. Taylor, M. R. Branco, Functional evaluation of transposable elements as enhancers in mouse embryonic and trophoblast stem cells. eLife 8 , e44344 (2019).31012843
24 M. Yu , Endogenous retrovirus-derived enhancers confer the transcriptional regulation of human trophoblast syncytialization. Nucl. Acids Res. 51 , 4745–4759 (2023), 10.1093/nar/gkad109.36864754
25 C. Du , Regulation of endogenous retrovirus-derived regulatory elements by GATA2/3 and MSX2 in human trophoblast stem cells. Genome Res. 33 , 197–207 (2023).36806146
26 J. M. Frost , Regulation of human trophoblast gene expression by endogenous retroviruses. Nat. Struct. Mol. Biol. 30 , 527–538 (2023), 10.1038/s41594-023-00960-6.37012406
27 J. Wang , Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. Nature 516 , 405–409 (2014).25317556
28 X. Lu , The retrovirus HERVH is a long noncoding RNA required for human embryonic stem cell identity. Nat. Struct. Mol. Biol. 21 , 423–425 (2014).24681886
29 S. Li , ESRG is critical to maintain the cell survival and self-renewal/pluripotency of hPSCs by collaborating with MCM2 to suppress p53 pathway. Int. J. Biol. Sci. 19 , 916–935 (2023).36778110
30 F. A. Santoni, J. Guerra, J. Luban, HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency. Retrovirology 9 , 111 (2012).23253934
31 M. Singh , A new human embryonic cell type associated with activity of young transposable elements allows definition of the inner cell mass. PLoS Biol. 21 , e3002162 (2023).37339119
32 T. A. Carter , Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo. eLife 11 , e76257 (2022).35179489
33 J. Goke , Dynamic transcription of distinct classes of endogenous retroviral elements marks specific populations of early human embryonic cells. Cell Stem Cell 16 , 135–141 (2015).25658370
34 Y. Zhang , Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells. Nat. Genet. 51 , 1380–1388 (2019).31427791
35 H. A. Lawson, Y. Liang, T. Wang, Transposable elements in mammalian chromatin organization. Nat. Rev. Genet. 24 , 712–723 (2023), 10.1038/s41576-023-00609-6.37286742
36 J. Wang, X. Lu, W. Zhang, G. H. Liu, Endogenous retroviruses in development and health. Trends Microbiol. (2023), 10.1016/j.tim.2023.09.006.
37 L. Liu , An integrated chromatin accessibility and transcriptome landscape of human pre-implantation embryos. Nat. Commun. 10 , 364 (2019).30664750
38 L. Xiang , A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577 , 537–542 (2020).31830756
39 S. Petropoulos , Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell 165 , 1012–1026 (2016).27062923
40 N. F. Hosseini, H. Manoochehri, S. G. Khoei, M. Sheykhhasan, The functional role of long non-coding RNA UCA1 in human multiple cancers: A review study. Curr. Mol. Med. 21 , 96–110 (2021).32560605
41 C. C. Sun , The functional role of LncRNA UCA1 in pancreatic cancer: A mini-review. J. Cancer 14 , 275–280 (2023).36741256
42 C. Apicella , Urothelial cancer associated 1 (UCA1) and miR-193 are two non-coding RNAs Involved in trophoblast fusion and placental diseases. Front. Cell Dev. Biol. 9 , 633937 (2021).34055770
43 J. Liu , Upregulated lncRNA UCA1 inhibits trophoblast cell invasion and proliferation by downregulating JAK2. J. Cell Physiol. 235 , 7410–7419 (2020).32067230
44 H. Shao , Urothelial carcinoma associated 1 promotes trophoblast invasion by regulating MMP9. Cell Biosci. 9 , 78 (2019).31572567
45 X. Xu, Y. Zhang, J. Li, B. Mao, Urothelial cancer associated 1 (UCA1) regulates trophoblast viability, proliferation, and migration via modulating the UCA1/miR-455/RUNX2 signaling pathway. Acta Biochim. Biophys. Sin. (Shanghai) 52 , 1120–1130 (2020).33085763
46 H. Okae , Derivation of human trophoblast stem cells. Cell Stem Cell 22 , 50–63.e56 (2018).29249463
47 S. Io , Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell 28 , 1023–1039.e13 (2021), 10.1016/j.stem.2021.03.013.33831365
48 A. Zani , Interferon-induced transmembrane proteins inhibit cell fusion mediated by trophoblast syncytins. J. Biol. Chem. 294 , 19844–19851 (2019).31735710
49 J. Buchrieser , IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise. Science 365 , 176–180 (2019).31296770
50 J. Ding , Mechanisms of immune regulation by the placenta: Role of type I interferon and interferon-stimulated genes signaling during pregnancy. Immunol. Rev. 308 , 9–24 (2022).35306673
51 H. Wu , Zika virus targets human trophoblast stem cells and prevents syncytialization in placental trophoblast organoids. Nat. Commun. 14 , 5541 (2023).37684223
52 S. A. Degrelle , IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases. iScience 26 , 107147 (2023).37434700
53 L. R. Jimi, M. Michael, M. Adithi, C. Lucia, L. C. Shawn, Investigation of human endogenous retrovirus-K (ERVK) expression and function in normal placentation and preterm pregnancy. bioRxiv [Preprint] (2021). 10.1101/2021.11.22.469425 (Accessed 19 October 2023).
54 P. A. Bolze, M. Mommert, F. Mallet, Contribution of syncytins and other endogenous retroviral envelopes to human placenta pathologies. Prog. Mol. Biol. Transl. Sci. 145 , 111–162 (2017).28110749
55 Z. Awamleh, G. B. Gloor, V. K. M. Han, Placental microRNAs in pregnancies with early onset intrauterine growth restriction and preeclampsia: Potential impact on gene expression and pathophysiology. BMC Med. Genomics 12 , 91 (2019).31248403
56 S. Wu , Trophoblast exosomal UCA1 induces endothelial injury through the PFN1-RhoA/ROCK pathway in preeclampsia: A human-specific adaptive pathogenic mechanism. Oxid. Med. Cell Longev. 2022 , 2198923 (2022).36160709
57 C. W. G. Redman, A. C. Staff, J. M. Roberts, Syncytiotrophoblast stress in preeclampsia: The convergence point for multiple pathways. Am. J. Obstet. Gynecol. 226 , S907–S927 (2022).33546842
58 T. Awoyemi , Preeclampsia and syncytiotrophoblast membrane extracellular vesicles (STB-EVs). Clin. Sci. (Lond) 136 , 1793–1807 (2022).36511102
59 I. Admati , Two distinct molecular faces of preeclampsia revealed by single-cell transcriptomics. Med 4 , 687–709.e7 (2023), 10.1016/j.medj.2023.07.005.37572658
60 S. Sun , Endogenous retrovirus expression activates type-I interferon signaling in an experimental mouse model of mesothelioma development. Cancer Lett. 507 , 26–38 (2021).33713739
61 C. Q. E. Lee , What is trophoblast? A combination of criteria define human first-trimester trophoblast. Stem Cell Rep. 6 , 257–272 (2016).
62 L. J. Yockey , Type I interferons instigate fetal demise after Zika virus infection. Sci. Immunol. 3 , eaao1680 (2018).29305462
63 K. B. Chiappinelli , Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses. Cell 162 , 974–986 (2015).26317466
64 F. Wang, X. Li, X. Xie, L. Zhao, W. Chen, UCA1, a non-protein-coding RNA up-regulated in bladder carcinoma and embryo, influencing cell growth and promoting invasion. FEBS Lett. 582 , 1919–1927 (2008).18501714
65 M. Xue, W. Chen, X. Li, Urothelial cancer associated 1: A long noncoding RNA with a crucial role in cancer. J. Cancer Res. Clin. Oncol. 142 , 1407–1419 (2016).26341664
66 G. P. Wagner, A. Kshitiz, A. L. Dighe, The coevolution of placentation and cancer. Annu. Rev. Anim. Biosci. 10 , 259–279 (2022).34780249
67 Kshitiz , Evolution of placental invasion and cancer metastasis are causally linked. Nat. Ecol. Evol. 3 , 1743–1753 (2019).31768023
68 P. K. Lala, P. Nandi, A. Hadi, C. Halari, A crossroad between placental and tumor biology: What have we learnt? Placenta 116 , 12–30 (2021).33958236
69 X. Kong , Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development. The National Genomics Data Center. https://ngdc.cncb.ac.cn/. Deposited 1 November 2023.
