Profiling of Pluripotency Factors in Single Cells and Early Embryos.

Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.

Control of noncoding RNA production and histone levels by a 5' tRNA fragment.

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs," are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF-5' tRF-Gly-GCC, or tRF-GG-as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs-snoRNAs, scaRNAs, and snRNAs-that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3' UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5' tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.

Transgenerational Epigenetic Inheritance.

Inheritance of genomic DNA underlies the vast majority of biological inheritance, yet it has been clear for decades that additional epigenetic information can be passed on to future generations. Here, we review major model systems for transgenerational epigenetic inheritance via the germline in multicellular organisms. In addition to surveying examples of epivariation that may arise stochastically or in response to unknown stimuli, we also discuss the induction of heritable epigenetic changes by genetic or environmental perturbations. Mechanistically, we discuss the increasingly well-understood molecular pathways responsible for epigenetic inheritance, with a focus on the unusual features of the germline epigenome.

Transcriptional and epigenetic control of early life cell fate decisions.

PURPOSE OF REVIEW: Global epigenetic reprogramming of the parental genomes after fertilization ensures the establishment of genome organization permissive for cell specialization and differentiation during development. In this review, we highlight selected, well-characterized relationships between epigenetic factors and transcriptional cell fate regulators during the initial stages of mouse development. RECENT FINDINGS: Blastomeres of the mouse embryo are characterized by atypical and dynamic histone modification arrangements, noncoding RNAs and DNA methylation profiles. Moreover, asymmetries in epigenomic patterning between embryonic cells arise as early as the first cleavage, with potentially instructive roles during the first lineage allocations in the mouse embryo. Although it is widely appreciated that transcription factors and developmental signaling pathways play a crucial role in cell fate specification at the onset of development, it is increasingly clear that their function is tightly connected to the underlying epigenetic status of the embryonic cells in which they act. SUMMARY: Findings on the interplay between genetic, epigenetic and environmental factors during reprogramming and differentiation in the embryo are crucial for understanding the molecular underpinnings of disease processes, particularly tumorigenesis, which is characterized by global epigenetic rewiring and progressive loss of cellular identity.

Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.

The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.

How mammals pack their sperm: a variant matter.

Producing competent gametes is essential for transmitting genetic information throughout generations. Spermatogenesis is a unique example of rearrangements of genome packaging to ensure fertilization. After meiosis, spermatids undergo drastic morphological changes, perhaps the most dramatic ones occurring in their nuclei, including the transition into a protamine-packaged genome. In this issue of Genes & Development, Montellier and colleagues (pp. 1680-1692) shed new light on the molecular mechanisms regulating this transition by ascribing for the first time a function to a histone variant, TH2B, in the regulation of this process.

Association of MTHFR polymorphism, folic acid and vitamin B12 with serum homocysteine levels in pregnant women.

Homocysteine is known to be associated with adverse vascular and metabolic effects, as well as pregnancy complications. Its serum levels are influenced by the function of the enzyme methylenetetrahydrofolate reductase (MTHFR) and the dietary intake of folic acid, vitamin B12, and methionine. In this cross-sectional study, we investigated the association of genetic polymorphisms of the MTHFR gene with vitamin status in pregnant women during mandatory folic acid supplementation. The study included 102 pregnant women between 24 and 28 weeks of gestation who were attending regular outpatient examinations at the maternity clinic. Homocysteine, folic acid, vitamin B12 levels, and MTHFR gene polymorphisms (C677T and A1298C) were analyzed. Significant associations were found between vitamin B12 and folic acid levels with homocysteine (P < 0.001), with lower serum levels of these vitamins being associated with higher homocysteine levels. Surprisingly, there was no significant association between MTHFR genetic polymorphisms and serum homocysteine levels, likely attributed to the supplementation of folic acid and vitamin B12 in vitamin supplements for pregnant women, which counteracts the effect of the mutation. Remarkably, a high prevalence of MTHFR gene mutations was found, with the C677T polymorphism present in 56.9% and the A1298C polymorphism in 87.2% of pregnant women. These findings emphasize the importance of adequate folic acid and vitamin B12 intake during pregnancy to regulate homocysteine levels. Although the MTHFR gene mutations were highly prevalent, their influence on homocysteine levels in this population appears to be mitigated by vitamin supplementation. Further research is warranted to explore the impact of these mutations on other aspects of pregnancy outcomes. The trial is registrated at Clinicaltrail.gov (NCT04952324).

Mechanisms of transgenerational epigenetic inheritance: lessons from animal model organisms.

Epigenetic inheritance is a phenomenon whereby stochastic or signal-induced changes to parental germline epigenome modulate phenotypic output in one or more subsequent generations, independently of mutations in the genomic DNA. While the number of reported epigenetic inheritance phenomena across phyla is exponentially growing, much remains to be elucidated about their mechanistic underpinnings, and their significance for organismal homeostasis and adaptation. Here, we review the most recent epigenetic inheritance examples in animal models, outlining molecular details behind environmental sensing by the germline, and the functional relationships connecting epigenetic mechanisms and phenotypic traits after fertilization. We touch upon the experimental challenges associated with studying the scope of environmental input on phenotypic outcomes between generations. Finally, we discuss the implications of mechanistic findings from model organisms for the emergent examples of parental effects in human populations.

Protein phosphorylation in bacterial signal transduction.

BACKGROUND: Protein phosphorylation has emerged as one of the major post translational modifications in bacteria, involved in regulating a myriad of physiological processes. In a complex and dynamic system such as the bacterial cell, connectivity of its components accounts for a number of emergent properties. This article is part of a Special Issue entitled: Systems Biology of Microorganisms. SCOPE OF REVIEW: This review focuses on the implications of bacterial protein phosphorylation in cell signaling and regulation and highlights the connections and cross talk between various signaling pathways: bacterial two-component systems and serine/threonine kinases, but also the interference between phosphorylation and other post-translational modifications (methylation and acetylation). MAJOR CONCLUSIONS: Recent technical developments in high accuracy mass spectrometry have profoundly transformed proteomics, and today exhaustive site-specific phosphoproteomes are available for a number of bacterial species. Nevertheless, prediction of phosphorylation sites remains the main guide for many researchers, so we discuss the characteristics, limits and advantages of available phosphorylation predictors. GENERAL SIGNIFICANCE: The advent of quantitative phosphoproteomics has brought the field on the doorstep of systems biology, but a number of challenges remain before the bacterial phosphorylation networks can be efficiently modeled and their physiological role understood. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Extending resolution within a single imaging frame.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Deficient spermiogenesis in mice lacking Rlim.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo.

After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

PREVALENCE OF GESTATIONAL DIABETES IN THE SOUTHERN PART OF BOSNIA AND HERZEGOVINA.

BACKGROUND: The prevalence of gestational diabetes mellitus (GDM), as a complex problem in pregnancy, is increasing all over the world, but most noticeable in developing countries. AIMS: To estimate GDM prevalence and associated pregnancy features in the southern part of Bosnia and Herzegovina. METHODS: A cross-sectional observational study was conducted from October 2010 through March 2011. A total of 285 pregnant women with singleton pregnancies participated and were asigned to the study in the order they came for their usual ante-natal clinic examination. They underwent an oral glucose tolerance test (OGTT) with 75 g of glucose. Information on OGTT results, maternal characteristics and pregnancy outcomes were collected from database and medical records. RESULTS: Prevalence of GDM was 10.9% according to 1999 World Health Organisation (WHO) diagnostic criteria. Prenatal cigarette smoking, previous GDM, cesarean delivery rate and neonatal hypoglycemia were significantly more frequent in the GDM group compared to the group of pregnancies with normal glucose tolerance (p = 0.015, p < 0.001, p = 0.015, p = 0.002). CONCLUSION: This study presents a relatively high prevalence of GDM in Bosnia and Herzegovina. There is a need for large well-designed study on GDM prevalence and its other features.

Regulation of X-linked gene expression during early mouse development by Rlim.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly.

Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced reprogramming to pluripotency is well established, totipotent cells remain poorly characterized, and whether reprogramming to totipotency is possible is unknown. We show that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1. Endogenous retroviruses and genes specific to 2-cell embryos are the highest-upregulated genes upon CAF-1 knockdown. Emerging 2C-like cells exhibit molecular characteristics of 2-cell embryos and higher reprogrammability than ES cells upon nuclear transfer. Our results suggest that early embryonic-like cells can be induced by modulating chromatin assembly and that atypical histone deposition may trigger the emergence of totipotent cells.

Evolution of bacterial protein-tyrosine kinases and their relaxed specificity toward substrates.

It has often been speculated that bacterial protein-tyrosine kinases (BY-kinases) evolve rapidly and maintain relaxed substrate specificity to quickly adopt new substrates when evolutionary pressure in that direction arises. Here, we report a phylogenomic and biochemical analysis of BY-kinases, and their relationship to substrates aimed to validate this hypothesis. Our results suggest that BY-kinases are ubiquitously distributed in bacterial phyla and underwent a complex evolutionary history, affected considerably by gene duplications and horizontal gene transfer events. This is consistent with the fact that the BY-kinase sequences represent a high level of substitution saturation and have a higher evolutionary rate compared with other bacterial genes. On the basis of similarity networks, we could classify BY kinases into three main groups with 14 subgroups. Extensive sequence conservation was observed only around the three canonical Walker motifs, whereas unique signatures proposed the functional speciation and diversification within some subgroups. The relationship between BY-kinases and their substrates was analyzed using a ubiquitous substrate (Ugd) and some Firmicute-specific substrates (YvyG and YjoA) from Bacillus subtilis. No evidence of coevolution between kinases and substrates at the sequence level was found. Seven BY-kinases, including well-characterized and previously uncharacterized ones, were used for experimental studies. Most of the tested kinases were able to phosphorylate substrates from B. subtilis (Ugd, YvyG, and YjoA), despite originating from very distant bacteria. Our results are consistent with the hypothesis that BY-kinases have evolved relaxed substrate specificity and are probably maintained as rapidly evolving platforms for adopting new substrates.

Analysis of active chromatin modifications in early mammalian embryos reveals uncoupling of H2A.Z acetylation and H3K36 trimethylation from embryonic genome activation.

Early embryonic development is characterized by dramatic changes in cell potency and chromatin organization. The role of histone variants in the context of chromatin remodeling during embryogenesis remains under investigated. In particular, the nuclear distribution of the histone variant H2A.Z and its modifications have not been examined. Here we investigated the dynamics of acetylation of H2A.Z and two other active chromatin marks, H3K9ac and H3K36me3, throughout murine and bovine pre-implantation development. We show that H2A.Z distribution is dynamic during the earliest stages of mouse development, with protein levels significantly varying across stages and lowest at the 2-cell stage. When present, H2A.Z localizes preferentially to euchromatin at all stages analyzed. H2A.Z is acetylated in pre-implantation blastomeres and is preferentially localized to euchromatin, in line with the known role of H2A.Zac in transcriptional activation. Interestingly, however, H2A.Zac is undetectable in mouse embryos at the 2-cell stage, the time of major embryonic genome activation (EGA). Similarly, H3K36me3 is present exclusively in the maternal chromatin immediately after fertilization but becomes undetectable in interphase nuclei at the 2-cell stage, suggesting uncoupling of these active marks with global embryonic transcription activation. In bovine embryos, which undergo EGA at the 8-cell stage, H2A.Zac can be detected in zygotes, 4-, 8- and 16-cell stage embryos as well as in blastocysts, indicating that the dynamics of H2A.Zac is not conserved in mammals. In contrast, H3K36me3 displays mostly undetectable and heterogeneous localization pattern throughout bovine pre-implantation development. Thus, our results suggest that 'canonical' active chromatin marks exhibit a dynamic behavior in embryonic nuclei, which is both stage- and species-specific. We hypothesize that chromatin of early embryonic nuclei is subject to fine-tuning through differential acquisition of histone marks, allowing for proper chromatin remodeling and developmental progression in a species-specific fashion.