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

Genome-wide analysis of transposon and retroviral insertions reveals preferential integrations in regions of DNA flexibility

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germline transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish, with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence integration of heterologous DNA in genomes, and have implications for targeted genome engineering

Dynamic chromatin remodeling in cycling human endometrium at single-cell level

Estrogen-dependent proliferation followed by progesterone-dependent differentiation of the endometrium culminates in a short implantation window. We performed single-cell assay for transposase-accessible chromatin with sequencing on endometrial samples obtained across the menstrual cycle to investigate the regulation of temporal gene networks that control embryo implantation. We identify uniquely accessible chromatin regions in all major cellular constituents of the endometrium, delineate temporal patterns of coordinated chromatin remodeling in epithelial and stromal cells, and gain mechanistic insights into the emergence of a receptive state through integrated analysis of enriched transcription factor (TF) binding sites in dynamic chromatin regions, chromatin immunoprecipitation sequencing analyses, and gene expression data. We demonstrate that the implantation window coincides with pervasive cooption of transposable elements (TEs) into the regulatory chromatin landscape of decidualizing cells and expression of TE-derived transcripts in a spatially defined manner. Our data constitute a comprehensive map of the chromatin changes that control TF activities in a cycling endometrium at cellular resolution

EndoTime: non-categorical timing estimates for luteal endometrium

STUDY QUESTION Can the accuracy of timing of luteal phase endometrial biopsies based on urinary ovulation testing be improved by measuring the expression of a small number of genes and a continuous, non-categorical modelling approach? SUMMARY ANSWER Measuring the expression levels of six genes (IL2RB, IGFBP1, CXCL14, DPP4, GPX3 and SLC15A2) is sufficient to obtain substantially more accurate timing estimates and to assess the reliability of timing estimates for each sample. WHAT IS KNOWN ALREADY Commercially available endometrial timing approaches based on gene expression require large gene sets and use a categorical approach that classifies samples as pre-receptive, receptive or post-receptive. STUDY DESIGN, SIZE, DURATION Gene expression was measured by RTq-PCR in different sample sets, comprising a total of 664 endometrial biopsies obtained 4–12 days after a self-reported positive home ovulation test. A further 36 endometrial samples were profiled by RTq-PCR as well as RNA-sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS A computational procedure, named ‘EndoTime’, was established that models the temporal profile of each gene and estimates the timing of each sample. Iterating these steps, temporal profiles are gradually refined as sample timings are being updated, and confidence in timing estimates is increased. After convergence, the method reports updated timing estimates for each sample while preserving the overall distribution of time points. MAIN RESULTS AND THE ROLE OF CHANCE The Wilcoxon rank-sum test was used to confirm that ordering samples by EndoTime estimates yields sharper temporal expression profiles for held-out genes (not used when determining sample timings) than ordering the same expression values by patient-reported times (GPX3: P  0.05). LARGE SCALE DATA The RTq-PCR data files are available via the GitHub repository for the EndoTime software at https://github.com/AE-Mitchell/EndoTime, as is the code used for pre-processing of RTq-PCR data. The RNA-sequencing data are available on GEO (accession GSE180485). LIMITATIONS, REASONS FOR CAUTION Timing estimates are informed by glandular gene expression and will only represent the temporal state of other endometrial cell types if in synchrony with the epithelium. Methods that estimate the day of ovulation are still required as these data are essential inputs in our method. Our approach, in its current iteration, performs batch correction such that larger sample batches impart greater accuracy to timing estimations. In theory, our method requires endometrial samples obtained at different days in the luteal phase. In practice, however, this is not a concern as timings based on urinary ovulation testing are associated with a sufficient level of noise to ensure that a variety of time points will be sampled. WIDER IMPLICATIONS OF THE FINDINGS Our method is the first to assay the temporal state of luteal-phase endometrial samples on a continuous domain. It is freely available with fully shared data and open-source software. EndoTime enables accurate temporal profiling of any gene in luteal endometrial samples for a wide range of research applications and, potentially, clinical use

Are psychiatrists trained to address the mental health needs of young people transitioning from child to adult services? Insights from a European survey

Background: In mental health, transition refers to the pathway of young people from child and adolescent to adult services. Training of mental health psychiatrists on transition-related topics offers the opportunity to improve clinical practice and experiences of young people reaching the upper age limit of child and adolescent care. Methods: National psychiatrist's organizations or experts from 21 European countries were surveyed 1/ to describe the status of transition in adult psychiatry (AP) and child and adolescent psychiatry (CAP) postgraduate training in Europe; 2/ to explore the amount of cross-training between both specialties. This survey was a part of the MILESTONE project aiming to study and improve the transition process of young people at the service boundary. Results: Transition was a mandatory topic in the AP curriculum of 1/19 countries (5%) and in the CAP curriculum of 4/17 countries (24%). Most topics relevant for transition planning were addressed during AP training in 7/17 countries (41%) to 10/17 countries (59%), and during CAP training in 9/11 countries (82%) to 13/13 countries (100%). Depending on the training models, theoretical education in CAP was mandatory during AP training in 94% (15/16) to 100% of the countries (3/3); and in AP during CAP training in 81% (13/16) to 100% of the countries (3/3). Placements were mandatory in CAP during AP training in 67% (2/3) to 71% of the countries (12/17); and in AP during CAP training in 87% (13/15) to 100% of the countries (3/3). Discussion and Conclusion: Specific training about transition is limited during CAP and AP postgraduate training in Europe. Cross-training between both specialties offers a basis for improved communication between child and adult services but efforts should be sustained in practical training. Recommendations are provided to foster further development and meet the specific needs of young people transitioning to adult services

Genotype-phenotype associations in WT1 glomerulopathy

WT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype-phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype-phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening.Kidney International advance online publication, 8 January 2014; doi:10.1038/ki.2013.519

A multifunctional mutagenesis system for analysis of gene function in zebrafish

10.1534/g3.114.015842G3: Genes, Genomes, Genetics561283-129