Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development.

The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.

CITED2 is a conserved regulator of the uterine-placental interface.

Establishment of the hemochorial uterine-placental interface requires exodus of trophoblast cells from the placenta and their transformative actions on the uterus, which represent processes critical for a successful pregnancy, but are poorly understood. We examined the involvement of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) in rat and human trophoblast cell development. The rat and human exhibit deep hemochorial placentation. CITED2 was distinctively expressed in the junctional zone (JZ) and invasive trophoblast cells of the rat. Homozygous Cited2 gene deletion resulted in placental and fetal growth restriction. Small Cited2 null placentas were characterized by disruptions in the JZ, delays in intrauterine trophoblast cell invasion, and compromised plasticity. In the human placentation site, CITED2 was uniquely expressed in the extravillous trophoblast (EVT) cell column and importantly contributed to the development of the EVT cell lineage. We conclude that CITED2 is a conserved regulator of deep hemochorial placentation.

Human trophoblast stem cells restrict human cytomegalovirus replication.

Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs), syncytiotrophoblasts (STBs), and organoids, and this study assessed the utility of TSCs as a model of HCMV infection in the first-trimester placenta. HCMV was found to non-productively infect TSCs, EVTs, and STBs. Immunofluorescence assays and flow cytometry experiments further revealed that infected TSCs frequently only express immediate early viral gene products. Similarly, RNA sequencing found that viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and Wingless/Integrated signaling. Thus, while HCMV does not replicate in TSCs, infection may perturb trophoblast differentiation in ways that could interfere with placental function. IMPORTANCE: Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

The multifaceted role of GCM1 during trophoblast differentiation in the human placenta.

Remodeling of the uterine vasculature by invasive extravillous trophoblasts (EVTs) is a critical aspect of human placentation. Insufficient EVT invasion can lead to severe obstetrical complications like preeclampsia, intrauterine growth restriction, and preterm birth. Glial cells missing-1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is highly expressed in human syncytiotrophoblast (ST) and EVTs. GCM1 is classically considered a master regulator of ST formation, but little is known about its contribution to the development and function of EVTs. Therefore, in this study we test the hypothesis that GCM1 is a critical regulator of both EVT and ST development and function. We show that GCM1 is highly expressed in human trophoblast stem (TS) cells differentiated into either ST or EVTs. Knockdown of GCM1 in TS cells hindered differentiation into both ST and EVT pathways. When placed in ST media, GCM1-knockdown cells formed small, unstable clusters; when placed in EVT media, cells had altered morphology and transcript profiles resembling cells trapped in an intermediate state between CT and EVT, and invasive capacity through matrix was reduced. RNA sequencing analysis of GCM1-deficient TS cells revealed downregulation of EVT-associated genes and enrichment in transcripts related to WNT signaling, which was linked to decreased expression of the EVT master regulator ASCL2 and the WNT antagonist NOTUM. Our findings reveal an essential role of GCM1 during ST and EVT development, and suggest that GCM1 regulates differentiation of human TS cells into EVTs by inducing expression of ASCL2 and NOTUM.

Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.

ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development.

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

Atypical protein kinase C iota (PKCλ/ι) ensures mammalian development by establishing the maternal-fetal exchange interface.

In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.

Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells.

A complete hydatidiform mole (CHM) is androgenetic in origin and characterized by enhanced trophoblastic proliferation and the absence of fetal tissue. In 15 to 20% of cases, CHMs are followed by malignant gestational trophoblastic neoplasms including choriocarcinoma. Aberrant genomic imprinting may be responsible for trophoblast hypertrophy in CHMs, but the detailed mechanisms are still elusive, partly due to the lack of suitable animal or in vitro models. We recently developed a culture system of human trophoblast stem (TS) cells. In this study, we apply this system to CHMs for a better understanding of their molecular pathology. CHM-derived TS cells, designated as TSmole cells, are morphologically similar to biparental TS (TSbip) cells and express TS-specific markers such as GATA3, KRT7, and TFAP2C. Interestingly, TSmole cells have a growth advantage over TSbip cells only after they reach confluence. We found that p57KIP2, a maternally expressed gene encoding a cyclin-dependent kinase inhibitor, is strongly induced by increased cell density in TSbip cells, but not in TSmole cells. Knockout and overexpression studies suggest that loss of p57KIP2 expression would be the major cause of the reduced sensitivity to contact inhibition in CHMs. Our findings shed light on the molecular mechanism underlying the pathogenesis of CHMs and could have broad implications in tumorigenesis beyond CHMs because silencing of p57KIP2 is frequently observed in a variety of human tumors.

Genomic imprinting in human placentation.

Background: Genomic imprinting (GI) is a mammalian-specific epigenetic phenomenon that has been implicated in the evolution of the placenta in mammals. Methods: Embryo transfer procedures and trophoblast stem (TS) cells were used to re-examine mouse placenta-specific GI genes. For the analysis of human GI genes, cytotrophoblast cells isolated from human placental tissues were used. Using human TS cells, the biological roles of human GI genes were examined. Main findings: (1) Many previously identified mouse GI genes were likely to be falsely identified due to contaminating maternal cells. (2) Human placenta-specific GI genes were comprehensively determined, highlighting incomplete erasure of germline DNA methylation in the human placenta. (3) Human TS cells retained normal GI patterns. (4) Complete hydatidiform mole-derived TS cells were characterized by aberrant GI and enhanced trophoblastic proliferation. The maternally expressed imprinted gene p57KIP2 may be responsible for the enhanced proliferation. (5) The primate-specific microRNA cluster on chromosome 19, which is a placenta-specific GI gene, is essential for self-renewal and differentiation of human TS cells. Conclusion: Genomic imprinting plays diverse and important roles in human placentation. Experimental analyses using TS cells suggest that the GI maintenance is necessary for normal placental development in humans.

Abnormal cleavage is involved in the self-correction of bovine preimplantation embryos.

Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.

Cutting Edge: G Protein Subunit ß 1 Negatively Regulates NLRP3 Inflammasome Activation.

The NLRP3 inflammasome has important roles in the pathogenesis of various inflammatory diseases. However, the regulatory mechanisms of the NLRP3 inflammasome are not fully understood. In this study, we attempted to identify molecules that interact with NLRP3 upon its activation. We identified G protein subunit ß 1 (GNB1), a downstream molecule of G protein-coupled receptors (GPCRs), which regulates the NLRP3 inflammasome activation. GNB1 was physically associated with NLRP3 via the pyrin domain of NLRP3. Activation of the NLRP3 inflammasome was enhanced in GNB1-knockdown or GNB1-deficient murine macrophages, although a lack of GNB1 did not affect activation of the AIM2 inflammasome. ASC oligomerization induced by NLRP3 was enhanced by GNB1 deficiency. Conversely, NLRP3-dependent ASC oligomerization was inhibited by the overexpression of GNB1. This study indicates that GNB1 negatively regulates NLRP3 inflammasome activation by suppressing NLRP3-dependent ASC oligomerization, and it provides a regulatory mechanism of the NLRP3 inflammasome.

G protein subunit ß1 is an important mediator of the late stage of endochondral ossification.

G protein signaling plays important roles in skeletal development. G protein subunit ß1 (GNB1) is a component of the G protein complex and is associated with G protein signaling. In humans, GNB1 mutations cause global developmental and persistent growth delays and severe neurodevelopmental disability. Similarly, Gnb1-knockout (KO) mice display growth retardation with neural tube defects. These genetic studies raise the possibility that GNB1 regulates skeletal development. This study was designed to investigate the role of GNB1 in skeletal development using Gnb1-KO mice. Gnb1-KO mice showed dwarfism, shortening of limbs, and a decreased ossifying zone of long bones. In situ hybridization and RT-qPCR analyses revealed that Col10a1 and Mmp13 expression was reduced in long bones of Gnb1-KO mice, while Runx2, Osterix, Ihh, and Ppr expression levels were similar to those in wild-type littermates. Gnb1-KO-derived osteoblasts maintained calcification abilities and the expression levels of osteoblast marker genes were unaltered, indicating that osteoblast differentiation and function were not affected in Gnb1-KO mice. Taken together, our results show that GNB1 is required for the late stage of endochondral bone formation by regulating Col10a1 and Mmp13 expression.

Unique features and emerging in vitro models of human placental development.

BACKGROUND: The placenta is an essential organ for the normal development of mammalian fetuses. Most of our knowledge on the molecular mechanisms of placental development has come from the analyses of mice, especially histopathological examination of knockout mice. Choriocarcinoma and immortalized cell lines have also been used for basic research on the human placenta. However, these cells are quite different from normal trophoblast cells. METHODS: In this review, we first provide an overview of mouse and human placental development with particular focus on the differences in the anatomy, transcription factor networks, and epigenetic characteristics between these species. Next, we discuss pregnancy complications associated with abnormal placentation. Finally, we introduce emerging in vitro models to study the human placenta, including human trophoblast stem (TS) cells, trophoblast and endometrium organoids, and artificial embryos. MAIN FINDINGS: The placental structure and development differ greatly between humans and mice. The recent establishment of human TS cells and trophoblast and endometrial organoids enhances our understanding of the mechanisms underlying human placental development. CONCLUSION: These in vitro models will greatly advance our understanding of human placental development and potentially contribute to the elucidation of the causes of infertility and other pregnancy complications.

Allele-Specific Methylome and Transcriptome Analysis Reveals Widespread Imprinting in the Human Placenta.

DNA methylation is globally reprogrammed after fertilization, and as a result, the parental genomes have similar DNA-methylation profiles after implantation except at the germline differentially methylated regions (gDMRs). We and others have previously shown that human blastocysts might contain thousands of transient maternally methylated gDMRs (transient mDMRs), whose maternal methylation is lost in embryonic tissues after implantation. In this study, we performed genome-wide allelic DNA methylation analyses of purified trophoblast cells from human placentas and, surprisingly, found that more than one-quarter of the transient-in-embryo mDMRs maintained their maternally biased DNA methylation. RNA-sequencing-based allelic expression analyses revealed that some of the placenta-specific mDMRs were associated with expression of imprinted genes (e.g., TIGAR, SLC4A7, PROSER2-AS1, and KLHDC10), and three imprinted gene clusters were identified. This approach also identified some X-linked gDMRs. Comparisons of the data with those from other mammals revealed that genomic imprinting in the placenta is highly variable. These findings highlight the incomplete erasure of germline DNA methylation in the human placenta; understanding this erasure is important for understanding normal placental development and the pathogenesis of developmental disorders with imprinting effects.

Genome-wide analysis of DNA methylation dynamics during early human development.

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

RNA sequencing-based identification of aberrant imprinting in cloned mice.

Animals cloned by somatic cell nuclear transfer (SCNT) provide a unique model for understanding the mechanisms of nuclear epigenetic reprogramming to a state of totipotency. Though many phenotypic abnormalities have been demonstrated in cloned animals, the underlying mechanisms are not well understood. In this study, we performed transcriptome-wide allelic expression analyses in brain and placental tissues of cloned mice. We found that Gab1, Sfmbt2 and Slc38a4 showed loss of imprinting in all cloned mice analyzed, which might be involved in placentomegaly of cloned mice. These three genes did not require de novo DNA methylation in growing oocytes for the establishment of imprinting, implying the involvement of a de novo DNA methylation-independent mechanism. Loss of Dlk1-Dio3 imprinting was also observed in nearly half of cloned mouse embryos and showed a strong correlation with embryonic lethality. Our findings are essential to understand the underlying mechanisms of developmental abnormalities of cloned animals. We also emphasize that particular attention should be paid to specific imprinted genes for therapeutic and agricultural applications of SCNT.

Re-investigation and RNA sequencing-based identification of genes with placenta-specific imprinted expression.

Within the vertebrate groups, only mammals are subject to a specialized epigenetic process termed genomic imprinting in which genes are preferentially expressed from one parental allele. Imprinted expression has been reported for >100 mouse genes and, for approximately one-quarter of these genes, the imprinted expression is specific to the placenta (or extraembryonic tissues). This seemingly placenta-specific imprinted expression has garnered much attention, as has the apparent lack of conserved imprinting between the human and mouse placenta. In this study, we used a novel approach to re-investigate the placenta-specific expression using embryo transfer and trophoblast stem cells. We analyzed 20 genes previously reported to show maternal allele-specific expression in the placenta, and only 8 genes were confirmed to be imprinted. Other genes were likely to be falsely identified as imprinted due to their relatively high expression in contaminating maternal cells. Next, we performed a genome-wide transcriptome assay and identified 133 and 955 candidate imprinted genes with paternal allele- and maternal allele-specific expression. Of those we analyzed in detail, 1/6 (Gab1) of the candidates for paternal allele-specific expression and only 1/269 (Ano1) candidates for maternal allele-specific expression were authentically imprinted genes. Imprinting of Ano1 and Gab1 was specific to the placenta and neither gene displayed allele-specific promoter DNA methylation. Imprinting of ANO1, but not GAB1, was conserved in the human placenta. Our findings impose a considerable revision of the current views of placental imprinting.

Imprinting methylation errors in ART.

There has been an increase in incidence reports of rare imprinting disorders associated with assisted reproductive technology (ART). ART, including in vitro fertilization and intracytoplasmic sperm injections, is an important treatment for infertile people of reproductive age and increasingly produces children. The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that ART techniques themselves may predispose embryos to acquire imprinting errors and diseases. In this review, we note that the particular steps of ART may be prone to induction of imprinting methylation errors during gametogenesis, fertilization and early embryonic development. In addition, we explain imprint-associated diseases and their causes. Moreover, from a Japanese nationwide epidemiological study of imprint-associated diseases, we determine their associations with ART. Epigenetic studies will be required to understand the pathogenesis, ART-related risk factor(s) and what precautions can be taken to prevent the occurrence of input methylation errors. We hope that the constitution of children born after each ART procedure will reveal the safest and most ethical approach to use, which will be invaluable for the future development of standard ART.

Polycomb Repressive Complex 1.1 Component, BCOR, Promotes Syncytiotrophoblast Differentiation in Mice and Humans.

Early defects in placenta development are thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. Differentiating trophoblast stem cells undergo a choreographed allocation of syncytiotrophoblast and extravillous trophoblast cells in response to signaling cues from the developing fetus and the uterine environment. The expression and activity of transcription factors and chromatin modifying enzymes change during differentiation to appropriately reshape the chromatin landscape in each cell type. We have previously found in mice that extraembryonic loss of BCOR, a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1 (PRC1.1), leads to a reduced labyrinth and expanded trophoblast giant cell population in the placenta. Molecular analysis of wild-type and BCOR loss-of-function male and female placentas by RNA-seq identified gene expression changes as early as E6.5. We found that BCOR is required to down regulate stem cell genes and repress factors that promote alternate lineages which leads to reduced levels of syncytiotrophoblasts. ChIP-seq experiments identified a number of directly bound functional targets including Pdgfa and Wnt7b . In humans, BCOR is mutated in X-linked syndromes involving fetal growth restriction and females with a heterozygous null mutation in BCOR can experience recurrent miscarriages. To establish a direct role for BCOR in human placental development, we used CRISPR/Cas9 to knockout BCOR in male (CT29) and female (CT30) human trophoblast stem cells. Mutant cell lines retained capacity for induced differentiation into syncytiotrophoblast and extravillous trophoblasts and exhibited minimal changes in gene expression. However, in 3D cell culture using trophoblast organoid media, BCOR knockout lines had significantly altered gene expression including homologs of stem cell genes upregulated in Bcor knockout mice. CUT&RUN experiments in self-renewing and 3D cell culture identified genes directly bound by BCOR. Single cell profiling of wild type, knockout, and a P85L pathogenic knock-in BCOR mutation showed a reduced capacity to differentiate into syncytiotrophoblasts after four days of differentiation. Together, these results suggest that BCOR is a conserved regulator of trophoblast development that represses stem cell genes during differentiation and maintains lineage fidelity by repressing genes that promote alternate cell fates.