Reconstituted ovaries self-assemble without an ovarian surface epithelium.

Three-dimensional (3D) stem cell models of the ovary have the potential to benefit women's reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.

TET1 facilitates specification of early human lineages including germ cells.

Ten Eleven Translocation 1 (TET1) is a regulator of localized DNA demethylation through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). To examine DNA demethylation in human primordial germ cell-like cells (hPGCLCs) induced from human embryonic stem cells (hESCs), we performed bisulfite-assisted APOBEC coupled epigenetic sequencing (bACEseq) followed by integrated genomics analysis. Our data indicates that 5hmC enriches at hPGCLC-specific NANOG, SOX17 or TFAP2C binding sites on hPGCLC induction, and this is accompanied by localized DNA demethylation. Using CRISPR-Cas9, we show that deleting the catalytic domain of TET1 reduces hPGCLC competency when starting with hESC cultured on mouse embryonic fibroblasts, and this phenotype can be rescued after transitioning hESCs to defined media and a recombinant substrate. Taken together, our study demonstrates the importance of 5hmC in facilitating hPGCLC competency, and the role of hESC culture conditions in modulating this effect.

In vitro germ cell induction from fertile and infertile monozygotic twin research participants.

Human induced pluripotent stem cells (hiPSCs) enable reproductive diseases to be studied when the reproductive health of the participant is known. In this study, monozygotic (MZ) monoamniotic (MA) twins discordant for primary ovarian insufficiency (POI) consent to research to address the hypothesis that discordant POI is due to a shared primordial germ cell (PGC) progenitor pool. If this is the case, reprogramming the twin's skin cells to hiPSCs is expected to restore equivalent germ cell competency to the twins hiPSCs. Following reprogramming, the infertile MA twin's cells are capable of generating human PGC-like cells (hPGCLCs) and amniotic sac-like structures equivalent to her fertile twin sister. Using these hiPSCs together with genome sequencing, our data suggest that POI in the infertile twin is not due to a genetic barrier to amnion or germ cell formation and support the hypothesis that during gestation, amniotic PGCs are likely disproportionately allocated to the fertile twin with embryo splitting.

EED is required for mouse primordial germ cell differentiation in the embryonic gonad.

Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis.

FGFR3 is expressed by human primordial germ cells and is repressed after meiotic initiation to form primordial oocytes.

Human germ cell development is a highly regulated process beginning soon after embryo implantation with the specification of primordial germ cells (PGCs) and ending in adulthood with the differentiation of gametes. Here, we show that fibroblast growth factor receptor 3 (FGFR3) is expressed by human PGCs during the first and second trimester, becoming repressed as PGCs differentiate into primordial oocytes. Using fluorescence-activated cell sorting (FACS) with antibodies that recognize FGFR3 followed by single-cell RNA sequencing, we show that isolating FGFR3-positive cells enriches for human PGCs. Taken together, FGFR3 could be used in future studies as a strategy to identify maturing PGCs in vitro.

Studying Annelida Regeneration in a Novel Model Organism: The Freshwater Aeolosoma viride.

Aeolosoma viride, a globally distributed freshwater annelid, has a semitransparent appearance with 10 to 12 segments, about 2 to 3 mm in length. It is easy to raise and handle in laboratory conditions. Due to its robust regenerative capacity and applicability of various molecular tools including EdU labeling, whole-mount in situ hybridization (WISH), and RNA interference (RNAi), it rises as a promising model for studying whole-body regeneration.

Human reproduction is regulated by retrotransposons derived from ancient Hominidae-specific viral infections.

Germ cells are essential to pass DNA from one generation to the next. In human reproduction, germ cell development begins with the specification of primordial germ cells (PGCs) and a failure to specify PGCs leads to human infertility. Recent studies have revealed that the transcription factor network required for PGC specification has diverged in mammals, and this has a significant impact on our understanding of human reproduction. Here, we reveal that the Hominidae-specific Transposable Elements (TEs) LTR5Hs, may serve as TEENhancers (TE Embedded eNhancers) to facilitate PGC specification. LTR5Hs TEENhancers become transcriptionally active during PGC specification both in vivo and in vitro with epigenetic reprogramming leading to increased chromatin accessibility, localized DNA demethylation, enrichment of H3K27ac, and occupation of key hPGC transcription factors. Inactivation of LTR5Hs TEENhancers with KRAB mediated CRISPRi has a significant impact on germ cell specification. In summary, our data reveals the essential role of Hominidae-specific LTR5Hs TEENhancers in human germ cell development.

Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)-the master regulator of XCI-which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.

Double triage to identify poorly annotated genes in maize: The missing link in community curation.

The sophistication of gene prediction algorithms and the abundance of RNA-based evidence for the maize genome may suggest that manual curation of gene models is no longer necessary. However, quality metrics generated by the MAKER-P gene annotation pipeline identified 17,225 of 130,330 (13%) protein-coding transcripts in the B73 Reference Genome V4 gene set with models of low concordance to available biological evidence. Working with eight graduate students, we used the Apollo annotation editor to curate 86 transcript models flagged by quality metrics and a complimentary method using the Gramene gene tree visualizer. All of the triaged models had significant errors-including missing or extra exons, non-canonical splice sites, and incorrect UTRs. A correct transcript model existed for about 60% of genes (or transcripts) flagged by quality metrics; we attribute this to the convention of elevating the transcript with the longest coding sequence (CDS) to the canonical, or first, position. The remaining 40% of flagged genes resulted in novel annotations and represent a manual curation space of about 10% of the maize genome (~4,000 protein-coding genes). MAKER-P metrics have a specificity of 100%, and a sensitivity of 85%; the gene tree visualizer has a specificity of 100%. Together with the Apollo graphical editor, our double triage provides an infrastructure to support the community curation of eukaryotic genomes by scientists, students, and potentially even citizen scientists.

Global impacts of chromosomal imbalance on gene expression in Arabidopsis and other taxa.

Changes in dosage of part of the genome (aneuploidy) have long been known to produce much more severe phenotypic consequences than changes in the number of whole genomes (ploidy). To examine the basis of these differences, global gene expression in mature leaf tissue for all five trisomies and in diploids, triploids, and tetraploids of Arabidopsis thaliana was studied. The trisomies displayed a greater spread of expression modulation than the ploidy series. In general, expression of genes on the varied chromosome ranged from compensation to dosage effect, whereas genes from the remainder of the genome ranged from no effect to reduced expression approaching the inverse level of chromosomal imbalance (2/3). Genome-wide DNA methylation was examined in each genotype and found to shift most prominently with trisomy 4 but otherwise exhibited little change, indicating that genetic imbalance is generally mechanistically unrelated to DNA methylation. Independent analysis of gene functional classes demonstrated that ribosomal, proteasomal, and gene body methylated genes were less modulated compared with all classes of genes, whereas transcription factors, signal transduction components, and organelle-targeted protein genes were more tightly inversely affected. Comparing transcription factors and their targets in the trisomies and in expression networks revealed considerable discordance, illustrating that altered regulatory stoichiometry is a major contributor to genetic imbalance. Reanalysis of published data on gene expression in disomic yeast and trisomic mouse cells detected similar stoichiometric effects across broad phylogenetic taxa, and indicated that these effects reflect normal gene regulatory processes.

Reduced Representation Bisulfite Sequencing in Maize.

DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution ( Cokus et al., 2008 ). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies ( Smith et al., 2009 ). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants ( Hsu et al., 2017 ). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.

Prenatal Growth Patterns and Birthweight Are Associated With Differential DNA Methylation and Gene Expression of Cardiometabolic Risk Genes in Human Placentas: A Discovery-Based Approach.

Inherent genetic programming and environmental factors affect fetal growth in utero. Epidemiologic data in growth-altered fetuses, either intrauterine growth restricted (IUGR) or large for gestational age (LGA), demonstrate that these newborns are at increased risk of cardiometabolic disease in adulthood. There is growing evidence that the in utero environment leads to epigenetic modification, contributing to eventual risk of developing heart disease or diabetes. In this study, we used reduced representation bisulfite sequencing to examine genome-wide DNA methylation variation in placental samples from offspring born IUGR, LGA, and appropriate for gestational age (AGA) and to identify differential methylation of genes important for conferring risk of cardiometabolic disease. We found that there were distinct methylation signatures for IUGR, LGA, and AGA groups and identified over 500 differentially methylated genes (DMGs) among these group comparisons. Functional and gene network analyses revealed expected relationships of DMGs to placental physiology and transport, but also identified novel pathways with biologic plausibility and potential clinical importance to cardiometabolic disease. Specific loci for DMGs of interest had methylation patterns that were strongly associated with anthropometric presentations. We further validated altered gene expression of these specific DMGs contributing to vascular and metabolic diseases (SLC36A1, PTPRN2, CASZ1, IL10), thereby establishing transcriptional effects toward assigning functional significance. Our results suggest that the gene expression and methylation state of the human placenta are related and sensitive to the intrauterine environment, as it affects fetal growth patterns. We speculate that these observed changes may affect risk for offspring in developing adult cardiometabolic disease.

Deubiquitinating Enzyme OTU5 Contributes to DNA Methylation Patterns and Is Critical for Phosphate Nutrition Signals.

Phosphate (Pi) starvation induces a suite of adaptive responses aimed at recalibrating cellular Pi homeostasis. Plants harboring a mutation in OVARIAN TUMOR DOMAIN-CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) showed altered DNA methylation of root hair-related genes and altered Pi-responsive root traits. Unlike the wild type, homozygous otu5 mutants did not respond to Pi starvation by increased lateral root formation and increased root hair length but formed very short root hairs when grown on low-Pi media. Under Pi-replete conditions, otu5 plants developed more root hairs than the wild type due to attenuated primary root growth, a phenotype that resembled that of Pi-deficient plants. Growth of plants on low-Pi media altered both H3K4 and H3K27 trimethylation levels at the transcriptional start site of a subset of genes encoding key players in Pi homeostasis, which was correlated with mRNA abundance changes of these genes. Pi starvation had a minor impact on DNA methylation. Differentially methylated regions were enriched in transposable elements, suggesting that DNA methylation associated with low Pi supply is required for maintaining genome integrity. It is concluded that DNA methylation and histone methylation constitute critical, interdependent regulatory components that orchestrate the activity of a subset of Pi-responsive genes.

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

BACKGROUND: Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. RESULTS: After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. CONCLUSION: Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

Optimized reduced representation bisulfite sequencing reveals tissue-specific mCHH islands in maize.

BACKGROUND: DNA methylation plays important roles in many regulatory processes in plants. It is economically infeasible to profile genome-wide DNA methylation at a single-base resolution in maize, given its genome size of ~2.5 Gb. As an alternative, we adapted region of interest (ROI)-directed reduced representation bisulfite sequencing (RRBS) to survey genome-wide methylation in maize. RESULTS: We developed a pipeline for selecting restriction enzymes in silico and experimentally showed that, in the maize genome, MseI- and CviQI-digested fragments are precisely enriched in promoters and gene bodies, respectively. We proceeded with comparisons of epigenomes and transcriptomes between shoots and tassels and found that the occurrences of highly methylated, tissue-specific, mCHH islands upstream of transcription start sites (TSSs) were positively correlated with differential gene expression. Furthermore, 5' regulatory regions between TSS and mCHH islands often contain putative binding sites of known transcription factors (TFs) that regulate the flowering process and the timing of the transition from the vegetative to the reproductive phase. By integrating MNase-seq and siRNA-seq data, we found that regions of mCHH islands accumulate 21nt-siRNAs in a tissue-specific manner, marking the transition to open chromatin, thereby ensuring the accessibility of TFs for tissue-specific gene regulation. CONCLUSIONS: Our ROI-directed RRBS pipeline is eminently applicable to DNA methylation profiling of large genomes. Our results provide novel insights into the tissue-specific epigenomic landscapes in maize, demonstrating that DNA methylation and siRNA and chromatin accessibility constitute a critical, interdependent component that orchestrates the transition from the vegetative to the reproductive phase.

H2B ubiquitylation and the histone chaperone Asf1 cooperatively mediate the formation and maintenance of heterochromatin silencing.

Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin factors results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication and repair by anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications that modify nucleosome dynamics, such as H2B ubiquitylation (H2Bub). We report here that H2Bub and Asf1 cooperatively promote transcriptional silencing at yeast telomeres and mating loci. Through real time monitoring of HML (Hidden MAT Left) locus silencing, we found that transcriptional repression was slowly initiated and never fully established in mutants lacking both Asf1 and H2Bub. These findings are consistent with impaired HML silencer-binding and spreading of repressor proteins, Sir2 and Sir3. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing at mating loci. Thus, the interplay between H2Hbub and Asf1 may fine-tune nucleosome dynamics and SIR protein recruitment, and represent an ongoing requirement for proper formation and maintenance of heterochromatin.

Profiling genome-wide DNA methylation.

DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

MethGo: a comprehensive tool for analyzing whole-genome bisulfite sequencing data.

BACKGROUND: DNA methylation is a major epigenetic modification regulating several biological processes. A standard approach to measure DNA methylation is bisulfite sequencing (BS-Seq). BS-Seq couples bisulfite conversion of DNA with next-generation sequencing to profile genome-wide DNA methylation at single base resolution. The analysis of BS-Seq data involves the use of customized aligners for mapping bisulfite converted reads and the bioinformatic pipelines for downstream data analysis. RESULTS: Here we developed MethGo, a software tool designed for the analysis of data from whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). MethGo provides both genomic and epigenomic analyses including: 1) coverage distribution of each cytosine; 2) global cytosine methylation level; 3) cytosine methylation level distribution; 4) cytosine methylation level of genomic elements; 5) chromosome-wide cytosine methylation level distribution; 6) Gene-centric cytosine methylation level; 7) cytosine methylation levels at transcription factor binding sites (TFBSs); 8) single nucleotide polymorphism (SNP) calling, and 9) copy number variation (CNV) calling. CONCLUSIONS: MethGo is a simple and effective tool for the analysis of BS-Seq data including both WGBS and RRBS. It contains 9 analyses in 5 major modules to profile (epi)genome. It profiles genome-wide DNA methylation in global and in gene level scale. It can also analyze the methylation pattern around the transcription factor binding sites, and assess genetic variations such as SNPs and CNVs. MethGo is coded in Python and is publically available at http://paoyangchen-laboratory.github.io/methgo/.