HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

Fetal liver development and implications for liver disease pathogenesis.

The metabolic, digestive and homeostatic roles of the liver are dependent on proper crosstalk and organization of hepatic cell lineages. These hepatic cell lineages are derived from their respective progenitors early in organogenesis in a spatiotemporally controlled manner, contributing to the liver's specialized and diverse microarchitecture. Advances in genomics, lineage tracing and microscopy have led to seminal discoveries in the past decade that have elucidated liver cell lineage hierarchies. In particular, single-cell genomics has enabled researchers to explore diversity within the liver, especially early in development when the application of bulk genomics was previously constrained due to the organ's small scale, resulting in low cell numbers. These discoveries have substantially advanced our understanding of cell differentiation trajectories, cell fate decisions, cell lineage plasticity and the signalling microenvironment underlying the formation of the liver. In addition, they have provided insights into the pathogenesis of liver disease and cancer, in which developmental processes participate in disease emergence and regeneration. Future work will focus on the translation of this knowledge to optimize in vitro models of liver development and fine-tune regenerative medicine strategies to treat liver disease. In this Review, we discuss the emergence of hepatic parenchymal and non-parenchymal cells, advances that have been made in in vitro modelling of liver development and draw parallels between developmental and pathological processes.

SOX9 reprograms endothelial cells by altering the chromatin landscape.

The transcription factor SOX9 is activated at the onset of endothelial-to-mesenchymal transition (EndMT) during embryonic development and in pathological conditions. Its roles in regulating these processes, however, are not clear. Using human umbilical vein endothelial cells (HUVECs) as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal genes and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 displays features of a pioneer transcription factor, such as opening of chromatin and leading to deposition of active histone modifications at silent chromatin regions, guided by SOX dimer motifs and H2A.Z enrichment. We further observe highly transient and dynamic SOX9 binding, possibly promoted through its eviction by histone phosphorylation. However, while SOX9 binding is dynamic, changes in the chromatin landscape and cell fate induced by SOX9 are persistent. Finally, our analysis of single-cell chromatin accessibility indicates that SOX9 opens chromatin to drive EndMT in atherosclerotic lesions in vivo. This study provides new insight into key molecular functions of SOX9 and mechanisms of EndMT and highlights the crucial developmental role of SOX9 and relevance to human disease.

Elucidating the importance and regulation of key enhancers for human MEIS1 expression.

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is a critical factor in the pathogenesis of a large subset of acute myeloid leukemia (AML). Despite the clinical relevance of MEIS1, its regulation is largely unknown. To understand the transcriptional regulatory mechanisms contributing to human MEIS1 expression, we created a knock-in green florescent protein (GFP) reporter system at the endogenous MEIS1 locus in a human AML cell line. Using this model, we have delineated and dissected a critical enhancer region of the MEIS1 locus for transcription factor (TF) binding through in silico prediction in combination with oligo pull-down, mass-spectrometry and knockout analysis leading to the identification of FLI1, an E-twenty-six (ETS) transcription factor, as an important regulator of MEIS1 transcription. We further show direct binding of FLI1 to the MEIS1 locus in human AML cell lines as well as enrichment of histone acetylation in MEIS1-high healthy and leukemic cells. We also observe a positive correlation between high FLI1 transcript levels and worse overall survival in AML patients. Our study expands the role of ETS factors in AML and our model constitutes a feasible tool for a more detailed understanding of transcriptional regulatory elements and their interactome.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

Signalling pathways and transcriptional regulators orchestrating liver development and cancer.

Liver development is controlled by key signals and transcription factors that drive cell proliferation, migration, differentiation and functional maturation. In the adult liver, cell maturity can be perturbed by genetic and environmental factors that disrupt hepatic identity and function. Developmental signals and fetal genetic programmes are often dysregulated or reactivated, leading to dedifferentiation and disease. Here, we highlight signalling pathways and transcriptional regulators that drive liver cell development and primary liver cancers. We also discuss emerging models derived from pluripotent stem cells, 3D organoids and bioengineering for improved studies of signalling pathways in liver cancer and regenerative medicine.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Repressive Epigenetic Signatures Safeguard the Liver.

To date, how epigenetic changes are regulated during liver regeneration remains unclear. In this issue of Developmental Cell, Wang and colleagues (2019) employed transcriptomic and epigenomic profiling to explore how Uhrf1, an epigenetic regulator of DNA methylation, functions in liver regeneration using a mouse model of partial hepatectomy.

Endothelial Sash1 Is Required for Lung Maturation through Nitric Oxide Signaling.

The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1-/- mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with ß-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.

The emergent landscape of the mouse gut endoderm at single-cell resolution.

Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points-before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories.

Hepatocyte Nuclear Factor 4-Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver.

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.

YAP transcriptionally regulates ErbB2 to promote liver cell proliferation.

The Hippo signaling pathway is implicated in regulation of liver size and dysregulation of this pathway contributes to tumorigenesis. The transcriptional targets and downstream pathways of the Hippo pathway effector YAP that contribute to liver growth have yet to be well-characterized. We examined the liver transcriptome in response to YAP overexpression and identify the ErbB signaling pathway as a mediator of cell growth downstream of YAP. ErbB2 is transcriptionally regulated by YAP in both the mouse liver and in HepG2 human hepatoma cells. Knockdown of YAP or pharmacological inhibition with verteporfin reduced ERBB2 levels in HepG2 cells. Analysis of ChIP-seq data revealed enrichment of the transcription factor TEAD4 at the ERBB2 promoter. Using luciferase reporter and chromatin immunoprecipitation assays, we show that YAP and TEAD4 directly bind to and activate a regulatory element in the ErbB2 promoter in both the mouse liver and HepG2 cells. Functionally, knockdown of YAP reduced EGF-induced ERBB2-mediated HepG2 cell proliferation and PI3K/AKT activation. Our findings highlight a mechanism by which YAP exerts its effects on liver cell proliferation through the ErbB signaling pathway by directly regulating the transcription of ErbB2.

S1P Stimulates Proliferation by Upregulating CTGF Expression through S1PR2-Mediated YAP Activation.

Dysregulation of the Hippo pathway in the liver results in overgrowth and eventually tumorigenesis. To date, several upstream mechanisms have been identified that affect the Hippo pathway, which ultimately regulate YAP, the major downstream effector of the pathway. However, upstream regulators of the Hippo pathway in the liver remain poorly defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been shown to stimulate hepatocellular carcinoma (HCC) cell proliferation, but whether the Hippo pathway is involved in S1P-stimulated HCC cell proliferation remains to be determined. Here it is demonstrated that S1P activates YAP and that the S1P receptor 2 (S1PR2/S1P2) mediates S1P-induced YAP activation in both human and mouse HCC cells. S1P promotes YAP-mediated upregulation of cysteine-rich protein 61 and connective tissue growth factor (CTGF), and stimulates HCC cell proliferation. By using siRNA-mediated knockdown approaches, only CTGF was required for S1P-stimulated cell proliferation. Of note, S1P activates YAP in a MST1/2-independent manner suggesting that the canonical Hippo kinase is not required for S1P-mediated proliferation in liver. The upregulation of CTGF and S1P2 were also observed in liver-specific YAP overexpression transgenic mouse hepatocytes. Moreover, YAP regulated liver differentiation-dependent gene expression by influencing the chromatin binding of HNF4α based on ChIP-seq analysis. Finally, results using gain- and loss-of-function approaches demonstrate that HNF4α negatively regulated S1P-induced CTGF expression.Implications: These findings reveal a role for S1P in stimulating HCC cell proliferation by upregulating CTGF expression through S1P2-mediated YAP activation. Mol Cancer Res; 16(10); 1543-55. ©2018 AACR.

Expression patterns of Yes-associated protein 1 in the developing mouse liver.

The Hippo signaling pathway regulates many cellular processes, but has been specifically associated with control organ size and tumor growth. Yes-associated protein 1 (YAP1) is a transcriptional cofactor, in the Hippo pathway, that regulates gene expression when localized in the nucleus. Elevated expression of YAP1 in adult mouse liver leads to hepatomegaly and can cause hepatocellular carcinoma; while the loss of function studies reveal its importance in regulating cholangiocyte development. Here, we report the expression of YAP1 in mouse embryonic and postnatal hepatic cells, using AFP-GFP transgenic mice to identify the hepatocyte lineage. At embryonic day (E) 8.5, YAP1 is highly expressed in the endoderm, but is not present in the nucleus. Between E9.5-12.5, hepatic cells display low levels of nuclear and non-nuclear YAP1. The nuclear expression of YAP1 is first detected in a small subset of hepatic cells starting at E13.5 when the hepatoblasts begin to differentiate into hepatocytes and cholangiocytes. At E18.5, nuclear YAP1 is nearly undetectable in hepatoblasts and hepatocytes, but enriched within the nuclei of cholangiocytes. These levels remain similar postnatally, consistent with the role of YAP1 in cholangiocyte specification and maintenance.

APELA promotes tumour growth and cell migration in ovarian cancer in a p53-dependent manner.

OBJECTIVE: APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. METHODS: The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. RESULTS: APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. CONCLUSIONS: Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA.

Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives.

Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.

The role of the innate immune response regulatory gene ABCF1 in mammalian embryogenesis and development.

ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis and arthritis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2). To study ABCF1 expression and function in development and disease, we used a single gene trap insertion in the Abcf1 gene in murine embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous Abcf1 promoter by following the expression of a ß-galactosidase reporter gene. From the ES cells, heterozygous mice (Abcf1+/-) were produced. No live born Abcf1-/- progeny were ever generated, and the lethality was not mouse strain-specific. Thus, we have determined that Abcf1 is an essential gene in development. Abcf1-/- mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while Abcf1+/- mice appeared developmentally normal. Abcf1+/- mice were fertile and showed no significant differences in their anatomy when compared with their wild type littermates. The Abcf1 promoter was found to be active in all organs in adult mice, but varies in levels of expression in specific cell types within tissues. Furthermore, we observed high promoter activity in the blastocysts and embryos. Overall, Abcf1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types.

A knock-in mouse strain facilitates dynamic tracking and enrichment of MEIS1.

Myeloid ecotropic viral integration site 1 (MEIS1), a HOX transcription cofactor, is a critical regulator of normal hematopoiesis, and its overexpression is implicated in a wide range of leukemias. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) gene-editing system, we generated a knock-in transgenic mouse line in which a green fluorescent protein (GFP) reporter and a hemagglutinin (HA) epitope tag are inserted near the translational start site of endogenous Meis1. This novel reporter strain readily enables tracking of MEIS1 expression at single-cell-level resolution via the fluorescence reporter GFP, and facilitates MEIS1 detection and purification via the HA epitope tag. This new Meis1 reporter mouse line provides powerful new approaches to track Meis1-expressing hematopoietic cells and to explore Meis1 function and regulation during normal and leukemic hematopoiesis.