The infiltration of human myometrium and cervix with leukocytes and the formation of a pro-inflammatory environment within the uterus have been associated with the initiation of both term and preterm parturition. The mechanism regulating the onset of this pro-inflammatory cascade is not fully elucidated. We demonstrate that prokineticin 1 (PROK1) is up-regulated in human myometrium and placenta during labor. The expression of PROK1 receptor remains unchanged during labor and is abundantly expressed in the myometrium. Gene array analysis identified 65 genes up-regulated by PROK1 in human myometrium, mainly cytokines and chemokines, including IL-1β, chemokine C-C motif ligand 3, and colony-stimulating factor 3. In addition, we demonstrate that PROK1 increases the expression of chemokine C-C motif ligand 20, IL-6, IL-8, prostaglandin synthase 2, and prostaglandin E(2) and F(2α) secretion. The treatment of myometrial explants with 100 ng/mL of lipopolysaccharide up-regulates the expression of PROK1, PROK1 receptor, and inflammatory mediators. The infection of myometrial explants with lentiviral microRNA targeting PROK1, preceding treatment with lipopolysaccharide, reduces the expression of inflammatory genes. We propose that PROK1 is a novel inflammatory mediator that can contribute to the onset of human parturition at term and partially mediate premature onset of inflammatory pathways during bacterial infection.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a 3-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using 3-dimensional primary tumour tissue explant cultures.

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.

An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF2α via the FP receptor.

Implantation requires communication between a receptive endometrium and a healthy blastocyst. This maternal-embryonic crosstalk involves local mediators within the uterine microenvironment. We demonstrate that a secreted protein, prokineticin 1 (PROK1), is expressed in the receptive endometrium and during early pregnancy. PROK1 induces expression of leukemia inhibitory factor (LIF) in endometrial epithelial cells and first trimester decidua via a Gq-Ca(2+)-cSrc-mitogen-activated protein kinase kinase-mediated pathway. We show that human embryonic chorionic gonadotropin (hCG) induces sequential mRNA expression of PROK1 and LIF in an in vivo baboon model, in human endometrial epithelial cells, and in first-trimester decidua. We have used micro RNA constructs targeted to PROK1 to demonstrate that hCG-mediated LIF expression in the endometrium is dependent on prior induction of PROK1. Dual immunohistochemical analysis colocalized expression of the luteinizing hormone/chorionic gonadotropin receptor, PROK1, PROKR1, and LIF to the glandular epithelial cells of the first trimester decidual tissue. PROK1 enhances adhesion of trophoblast cells to fibronectin and laminin matrices, which are mediated predominantly via LIF induction. These data describe a novel signaling pathway mediating maternal-embryonic crosstalk, in which embryonic hCG via endometrial PROK1 may play a pivotal role in enhancing receptivity and maintaining early pregnancy.

Epigenetic changes are being increasingly recognized as a prominent feature of cancer. This occurs not only at individual genes, but also over larger chromosomal domains. To investigate this, we set out to identify large chromosomal domains of epigenetic dysregulation in breast cancers.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulk-level patterns of DNA methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome.

DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20-150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely.

Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor, PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1-prokineticin receptor 1 (PROKR1) signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine interleukin (IL)-11. Here, we have shown that PROK1-PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium-calcineurin signalling pathway in a guanine nucleotide-binding protein (G(q/11)), extracellular signal-regulated kinases, Ca(2+) and calcineurin-nuclear factor of activated T cells dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11Ralpha and PROKR1 within the stromal and glandular epithelial cells of non-pregnant endometrium and first trimester decidua. Overall we have identified and characterized the signalling components of a novel PROK1-PROKR1 signalling pathway regulating IL-11.

Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures.

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that in cancer, aberrantly active LINE-1 promoters can drive transcription of flanking unique sequences giving rise to LINE-1 chimeric transcripts (LCTs). Here, we show that one such LCT, LCT13, is a large transcript (>300 kb) running antisense to the metastasis-suppressor gene TFPI-2. We have modelled antisense RNA expression at TFPI-2 in transgenic mouse embryonic stem (ES) cells and demonstrate that antisense RNA induces silencing and deposition of repressive histone modifications implying a causal link. Consistent with this, LCT13 expression in breast and colon cancer cell lines is associated with silencing and repressive chromatin at TFPI-2. Furthermore, we detected LCT13 transcripts in 56% of colorectal tumours exhibiting reduced TFPI-2 expression. Our findings implicate activation of LINE-1 elements in subsequent epigenetic remodelling of surrounding genes, thus hinting a novel retrotransposition-independent role for LINE-1 elements in malignancy.

The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. However, a crucial issue is whether this level of nuclear organisation directly affects gene function, rather than merely reflecting it. To directly investigate whether proximity to the nuclear periphery can influence gene expression in mammalian cells, here we relocate specific human chromosomes to the nuclear periphery by tethering them to a protein of the inner nuclear membrane. We show that this can reversibly suppress the expression of some endogenous human genes located near the tethering sites, and even genes further away. However, the expression of many other genes is not detectably reduced and we show that location at the nuclear periphery is not incompatible with active transcription. The dampening of gene expression around the nuclear periphery is dependent on the activity of histone deacetylases. Our data show that the radial position within the nucleus can influence the expression of some, but not all, genes. This is compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate the expression of selected genes during development and differentiation.

Parent-of-origin effects (POE) exist when there is differential expression of alleles inherited from the two parents. A genome-wide scan for POE on DNA methylation at 639,238 CpGs in 5,101 individuals identifies 733 independent methylation CpGs potentially influenced by POE at a false discovery rate ≤ 0.05 of which 331 had not previously been identified. Cis and trans methylation quantitative trait loci (mQTL) regulate methylation variation through POE at 54% (399/733) of the identified POE-influenced CpGs. The combined results provide strong evidence for previously unidentified POE-influenced CpGs at 171 independent loci. Methylation variation at 14 of the POE-influenced CpGs is associated with multiple metabolic traits. A phenome-wide association analysis using the POE mQTL SNPs identifies a previously unidentified imprinted locus associated with waist circumference. These results provide a high resolution population-level map for POE on DNA methylation sites, their local and distant regulators and potential consequences for complex traits.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.

Prokineticin-1 (PROK1) and connective tissue growth factor (CTGF) are expressed in human endometrium and first-trimester decidua and have individually been proposed to have roles in implantation and placentation. We have recently demonstrated that CTGF may be a target gene for PROK1 in gene array analysis of a prokineticin receptor-1 stably transfected Ishikawa endometrial epithelial cell line (PROKR1-Ishikawa). The first aim of the study was to determine the effect of PROK1 on CTGF expression in PROKR1-Ishikawa cells and first-trimester decidua samples. Secondly, the effect of CTGF on trophoblast-derived HTR-8/SVneo cell adhesion and network formation was investigated.

The conceptus and endometrium secrete large amounts of prostaglandin E₂ (PGE₂) into the porcine uterine lumen during the periimplantation period. We hypothesized that PGE₂ acts on conceptus/trophoblast cells through auto- and paracrine mechanisms. Real-time RT-PCR analysis revealed that PGE₂ receptor (PTGER)2 mRNA was 14-fold greater in conceptuses/trophoblasts on days 14-25 (implantation and early placentation period) vs preimplantation day 10-13 conceptuses (P < .05). Similarly, expression of PTGER2 protein increased during implantation. Conceptus expression of PTGER4 mRNA and protein did not differ on days 10-19. PGE₂ stimulated PTGER2 mRNA expression in day 15 trophoblast cells through PTGER2 receptor signaling. PGE₂ elevated aromatase expression and estradiol-17β secretion by trophoblast cells. Moreover, PGE₂ and the PTGER2 agonist, butaprost, increased the adhesive capacity of both human HTR-8/SVneo trophoblast and primary porcine trophoblast cells to extracellular matrix. This PGE₂-induced alteration in trophoblast cell adhesion to extracellular matrix was abolished by incubation of these cells with AH6809 (PTGER2 antagonist), ITGAVB3-directed tetrapeptide arg-gly-asp-ser or integrin ITGAVB3 antibody. PGE₂ stimulated adhesion of porcine trophoblast cells via the estrogen receptor and MEK/MAPK signaling pathway. PGE₂ induced phosphorylation of MAPK1/MAPK3 through PTGER2 and up-regulated expression of cell adhesion proteins such as focal adhesion kinase and intercellular adhesion molecule-1. Our study indicates that elevated PGE₂ in the periimplantation uterine lumen stimulates conceptus PTGER2 expression, which in turn promotes trophoblast adhesion via integrins, and synthesis and secretion of the porcine embryonic signal estradiol-17β. Moreover, the mechanism through which PGE₂ increases trophoblast adhesion is not species specific because it is PTGER2- and integrin-dependent in both porcine and human trophoblast cells.