Analysis of chromatin accessibility in decidualizing human endometrial stromal cells

Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif-basic leucine zipper-as part of a triple motif that also comprised the estrogen receptor and Pax domain binding sites. Without exception, triple motifs were located within Alu elements, which suggests a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally under-represented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression

Impact of sustained transforming growth factor-β receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-β receptor (TGFβ-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFβ-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFβ-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARβ). Selective RARβ inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture

Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage

The glycosyltransferase EOGT regulates adropin expression in decidualizing human endometrium

In pregnancy, resistance of endometrial decidual cells to stress signals is critical for the integrity of the feto-maternal interface and, by extension, survival of the conceptus. O-GlcNAcylation is an essential post-translational modification that links glucose sensing to cellular stress resistance. Unexpectedly, decidualization of primary endometrial stromal cells (EnSCs) was associated with a 60% reduction in O-GlcNAc modified proteins, reflecting downregulation of the enzyme that adds O-GlcNAc to substrates (O-GlcNAc transferase, OGT) but not the enzyme that removes the modification (O-GlcNAcase, OGA). Notably, EOGT, an endoplasmic reticulum-specific O-GlcNAc transferase that modifies a limited number of secreted and membrane proteins, was markedly induced in differentiating EnSCs. Knockdown of EOGT perturbed a network of decidual genes involved in multiple cellular functions. The most downregulated gene upon EOGT knockdown in decidualizing cells was ENHO, which encodes adropin, a metabolic hormone involved in energy homeostasis and glucose and fatty acid metabolism. Analysis of mid-luteal endometrial biopsies revealed an inverse correlation between endometrial EOGT and ENHO expression and body mass index. Taken together, our findings reveal that obesity impairs the EOGT-adropin axis in decidual cells, which in turn points towards a novel mechanistic link between metabolic disorders and adverse pregnancy outcome. [Abstract copyright: Copyright © 2017 Endocrine Society.

A multifunctional mutagenesis system for analysis of gene function in zebrafish

Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease

Characterization of highly proliferative decidual precursor cells during the window of implantation in human endometrium

Pregnancy depends on the wholesale transformation of the endometrium, a process driven by differentiation of endometrial stromal cells (EnSC) into specialist decidual cells. Upon embryo implantation, decidual cells impart the tissue plasticity needed to accommodate a rapidly growing conceptus and invading placenta, although the underlying mechanisms are unclear. Here we characterize a discrete population of highly proliferative mesenchymal cells (hPMC) in midluteal human endometrium, coinciding with the window of embryo implantation. Single-cell transcriptomics demonstrated that hPMC express genes involved in chemotaxis and vascular transmigration. Although distinct from resident EnSC, hPMC also express genes encoding pivotal decidual transcription factors and markers, most prominently prolactin. We further show that hPMC are enriched around spiral arterioles, scattered throughout the stroma, and occasionally present in glandular and luminal epithelium. The abundance of hPMC correlated with the in vitro colony-forming unit activity of midluteal endometrium and, conversely, clonogenic cells in culture express a gene signature partially conserved in hPMC. Cross-referencing of single-cell RNA-sequencing data sets indicated that hPMC differentiate into a recently discovered decidual subpopulation in early pregnancy. Finally, we demonstrate that recurrent pregnancy loss is associated with hPMC depletion. Collectively, our findings characterize midluteal hPMC as novel decidual precursors that are likely derived from circulating bone marrow-derived mesenchymal stem/stromal cells and integral to decidual plasticity in pregnancy

Role of Smads during renal branching morphogenesis

Transforming growth factor-p (TGFp) pathways have been shown to regulate renal branching morphogenesis, one of the key processes determining the final number of nephrons. Although Smads are known to be downstream mediators of these signals, little is known about their role in kidney development. Using RT-PCR and in situ hybridization we investigated the expression of Smads during kidney development. We have found that the receptor regulated Smads (1, 2, 3, 5 and 8) as well as the common partner (Smad4), are expressed in the developing kidney during nephrogenesis. These factors have a specific expression pattern that overlaps with the expression of the signalling molecules BMP-4 and TGFpi. Expression of Smads 1, 3, 4, 5 and 8 is mainly found in undifferentiated mesenchymal cells of the nephrogenic zone as well as ureteric bud tips, but is downregulated in condensing mesenchymal structures. Smad 2 is mostly expressed in the stromal cell compartment. The distinct, yet overlapping, expression of Smads suggests that the specificity of TGFp signals might be determined in part by the combination of Smads expressed in a particular cell type. To better characterize the role of TGFP in kidney development, mouse embryonic kidney explants were treated with soluble TGFP ligands. Addition of BMP-2 and TGFpi inhibited renal branching morphogenesis through their effects on proliferation and survival. Similarly, proliferation and survival appeared to be the critical processes affected by TGFP signalling in the mIMCD-3 model of tubulogenesis. The direct role of Smads was examined by overexpression of a Smad3-GFP fusion protein in mIMCD-3 cells. Apoptosis was most frequently co-localized to Smad-3 transfected cells, which was consistent with the effects observed from the ligand, TGFpi. To test these observations in vivo, we developed a novel method for the manipulation of embryonic kidney explants in culture. GFP-expression vectors were introduced in kidney explants without loss in viability, and the expression of GFP was found to be rapid and sustained for several days in culture. In summary, this study has placed Smads downstream of TGFP signals in the developing kidney, and it opens the door to finding molecules that might be affected by these signals

Enhanced differentiation potential of primary human endometrial cells cultured on 3D scaffolds

Novel approaches for culturing primary human cells in vitro are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Conventional 2D monolayer cultures of endometrial epithelial and stromal cells fail to replicate the complex 3D architecture of tissue. A fully synthetic scaffold that mimics the microenvironment of the human endometrium can ultimately provide a robust platform for investigating tissue physiology and, hence, take significant steps toward tackling female infertility and IVF failure. In this work, emulsion-templated porous polymers (known as polyHIPEs) were investigated as scaffolds for the culture of primary human endometrial epithelial and stromal cells (HEECs and HESCs). Infiltration of HEECs and HESCs into cell-seeded polyHIPE scaffolds was assessed by histological studies, and phenotype was confirmed by immunostaining. Confocal microscopy revealed that the morphology of HEECs and HESCs is representative of that found in vivo. RNA sequencing was used to investigate transcriptome differences between cells grown on polyHIPE scaffolds and in monolayer cultures. The differentiation status of HEECs and HESCs grown in polyHIPE scaffolds and in monolayer cultures was further evaluated by monitoring the expression of endometrial marker genes. Our observations suggest that a 3D cell culture model that could approximate native human endometrial architecture and function can be developed using tailored polyHIPE scaffolds