Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

BRCA1 and ELK-1 regulate neural progenitor cell fate in the optic tectum in response to visual experience in Xenopus laevis tadpoles.

In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we profiled the transcriptomes of proliferating neural progenitor cells and newly differentiated neurons using RNA-Seq. We used advanced bioinformatic analysis of 1,130 differentially expressed transcripts to identify six differentially regulated transcriptional regulators, including Breast Cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1, which are predicted to regulate the majority of the other differentially expressed transcripts. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the potential functions of BRCA1 and ELK-1 in activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis showed that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 and ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.

Early life exposure to perfluorooctanesulfonate (PFOS) impacts vital biological processes in Xenopus laevis: Integrated morphometric and transcriptomic analyses.

Perfluorooctanesulfonate (PFOS) is a ubiquitous environmental pollutant associated with increasing health concerns and environmental hazards. Toxicological analyses of PFOS exposure are hampered by large interspecies variations and limited studies on the mechanistic details of PFOS-induced toxicity. We investigated the effects of PFOS exposure on Xenopus laevis embryos based on the reported developmental effects in zebrafish. X. laevis was selected to further our understanding of interspecies variation in response to PFOS, and we built upon previous studies by including transcriptomics and an assessment of ciliogenic effects. Midblastula-stage X. laevis embryos were exposed to PFOS using the frog embryo teratogenesis assay Xenopus (FETAX). Results showed teratogenic effects of PFOS in a time- and dose-dependent manner. The morphological abnormalities of skeleton deformities, a small head, and a miscoiled gut were associated with changes in gene expression evidenced by whole-mount in situ hybridization and transcriptomics. The transcriptomic profile of PFOS-exposed embryos indicated the perturbation in the expression of genes associated with cell death, and downregulation in adenosine triphosphate (ATP) biosynthesis. Moreover, we observed the effects of PFOS exposure on cilia development as a reduction in the number of multiciliated cells and changes in the directionality and velocity of the cilia-driven flow. Collectively, these data broaden the molecular understanding of PFOS-induced developmental effects, whereby ciliary dysfunction and disrupted ATP synthesis are implicated as the probable modes of action of embryotoxicity. Furthermore, our findings present a new challenge to understand the links between PFOS-induced developmental toxicity and vital biological processes.

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus.

BACKGROUND: Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. RESULTS: Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. CONCLUSIONS: Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal's unique physiology or environmental adaptation.

regeneration factors expressed on myeloid expression in macrophage-like cells is required for tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles can regenerate whole tails after amputation. We have previously reported that interleukin 11 (il11) is required for tail regeneration. In this study, we have screened for genes that support tail regeneration under Il11 signaling in a certain cell type and have identified the previously uncharacterized genes Xetrov90002578m.L and Xetrov90002579m.S [referred to hereafter as regeneration factors expressed on myeloid.L (rfem.L) and rfem.S]. Knockdown (KD) of rfem.L and rfem.S causes defects of tail regeneration, indicating that rfem.L and/or rfem.S are required for tail regeneration. Single-cell RNA sequencing (scRNA-seq) revealed that rfem.L and rfem.S are expressed in a subset of leukocytes with a macrophage-like gene expression profile. KD of colony-stimulating factor 1 (csf1), which is essential for macrophage differentiation and survival, reduced rfem.L and rfem.S expression levels and the number of rfem.L- and rfem.S-expressing cells in the regeneration bud. Furthermore, forced expression of rfem.L under control of the mpeg1 promoter, which drives rfem.L in macrophage-like cells, rescues rfem.L and rfem.S KD-induced tail regeneration defects. Our findings suggest that rfem.L or rfem.S expression in macrophage-like cells is required for tail regeneration.

Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis.

Protein Phosphatase 4 Catalytic Subunit (PPP4C) is an evolutionarily conserved protein involved in multiple biological and pathological events, including embryogenesis, organogenesis, cellular homeostasis, and oncogenesis. However, the detailed mechanisms underlying these processes remain largely unknown. Thus, we investigated the potential correlation between PPP4C and biological processes (BPs) and canonical Wnt signaling using pan-cancer analysis and Xenopus laevis (X. laevis) embryo model. Our results indicate that PPP4C is a potential biomarker for specific cancer types due to its high diagnostic accuracy and significant prognostic correlation. Furthermore, in multiple cancer types, PPP4C-related differentially expressed genes (DEGs) were significantly enriched in pattern specification, morphogenesis, and canonical Wnt activation. Consistently, perturbation of Ppp4c in X. laevis embryos interfered with normal embryogenesis and canonical Wnt responses. Moreover, biochemical analysis of X. laevis embryos demonstrated that both endogenous and exogenous Ppp4c negatively regulated AXIN1 (Wnt inhibitor) abundance. This study provides novel insights into PPP4C roles in pattern specification and Wnt activation. The similarities in BPs and Wnt signaling regulation regarding PPP4C support the intrinsic link between tumorigenesis and early embryogenesis.

Activation of P53 pathway contributes to Xenopus hybrid inviability.

Hybrid incompatibility as a kind of reproductive isolation contributes to speciation. The nucleocytoplasmic incompatibility between Xenopus tropicalis eggs and Xenopus laevis sperm (te×ls) leads to specific loss of paternal chromosomes 3L and 4L. The hybrids die before gastrulation, of which the lethal causes remain largely unclear. Here, we show that the activation of the tumor suppressor protein P53 at late blastula stage contributes to this early lethality. We find that in stage 9 embryos, P53-binding motif is the most enriched one in the up-regulated Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) peaks between te×ls and wild-type X. tropicalis controls, which correlates with an abrupt stabilization of P53 protein in te×ls hybrids at stage 9. Inhibition of P53 activity via either tp53 knockout or overexpression of a dominant-negative P53 mutant or Murine double minute 2 proto-oncogene (Mdm2), a negative regulator of P53, by mRNA injection can rescue the te×ls early lethality. Our results suggest a causal function of P53 on hybrid lethality prior to gastrulation.

RAF1 deficiency causes a lethal syndrome that underscores RTK signaling during embryogenesis.

Somatic and germline gain-of-function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor-prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss-of-function of the proto-oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress-induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Polyploidy in Xenopus lowers metabolic rate by decreasing total cell surface area.

Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here, we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na+/K+ ATPase, reducing energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism.

Modification of maternally defined H3K4me3 regulates the inviability of interspecific Xenopus hybrids.

Increasing evidence suggests that interspecific hybridization is crucial to speciation. However, chromatin incompatibility during interspecific hybridization often renders this process. Genomic imbalances such as chromosomal DNA loss and rearrangements leading to infertility have been commonly noted in hybrids. The mechanism underlying reproductive isolation of interspecific hybridization remains elusive. Here, we identified that modification of maternally defined H3K4me3 in Xenopus laevis and Xenopus tropicalis hybrids determines the different fates of the two types of hybrids as te×ls with developmental arrest and viable le×ts. Transcriptomics highlighted that the P53 pathway was overactivated, and the Wnt signaling pathway was suppressed in te×ls hybrids. Moreover, the lack of maternal H3K4me3 in te×ls disturbed the balance of gene expression between the L and S subgenomes in this hybrid. Attenuation of p53 can postpone the arrested development of te×ls. Our study suggests an additional model of reproductive isolation based on modifications of maternally defined H3K4me3.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Population genomics and subgenome evolution of the allotetraploid frog Xenopus laevis in southern Africa.

Allotetraploid genomes have two distinct genomic components called subgenomes that are derived from separate diploid ancestral species. Many genomic characteristics such as gene function, expression, recombination, and transposable element mobility may differ significantly between subgenomes. To explore the possibility that subgenome population structure and gene flow may differ as well, we examined genetic variation in an allotetraploid frog-the African clawed frog (Xenopus laevis)-over the dynamic and varied habitat of its native range in southern Africa. Using reduced representation genome sequences from 91 samples from 12 localities, we found no strong evidence that population structure and gene flow differed substantially by subgenome. We then compared patterns of population structure in the nuclear genome to the mitochondrial genome using Sanger sequences from 455 samples from 183 localities. Our results provide further resolution to the geographic distribution of mitochondrial and nuclear diversity in this species and illustrate that population structure in both genomes corresponds roughly with variation in seasonal rainfall and with the topography of southern Africa.

Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

Evolution and Potential Subfunctionalization of Duplicated fms-Related Class III Receptor Tyrosine Kinase flt3s and Their Ligands in the Allotetraploid Xenopus laevis.

The fms-related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulators of hematopoiesis and dendritic cell (DC) homeostasis with unsettled coevolution. Gene synteny and deduced amino acid sequence analyses identified conserved flt3 gene orthologs across all jawed vertebrates. In contrast, flt3lg orthologs were not retrieved in ray-finned fish, and the gene locus exhibited more variability among species. Interestingly, duplicated flt3/flt3lg genes were maintained in the allotetraploid Xenopus laevis Comparison of modeled structures of X. laevis Flt3 and Flt3lg homoeologs with the related diploid Xenopus tropicalis and with humans indicated a higher conformational divergence between the homoeologous pairs than their respective counterparts. The distinctive developmental and tissue expression patterns of Flt3 and Flt3lg homoeologs in tadpoles and adult frogs suggest a subfunctionalization of these homoeologs. To characterize Flt3 cell surface expression, X. laevis-tagged rFlt3lg.S and rFlt3lg.L were produced. Both rFlt3lg.S and rFlt3lg.L bind in vitro Flt3.S and Flt3.L and can trigger Erk1/2 signaling, which is consistent with a partial overlapping function between homoeologs. In spleen, Flt3.S/L cell surface expression was detected on a fraction of B cells and a population of MHC class IIhigh/CD8+ leukocytes phenotypically similar to the recently described dual follicular/conventional DC-like XL cells. Our result suggests that 1) Flt3lg.S and Flt3lg.L are both involved in XL cell homeostasis and that 2) XL cells have hematopoietic origin. Furthermore, we detected surface expression of the macrophage/monocyte marker Csf1r.S on XL cells as in mammalian and chicken DCs, which points to a common evolutionary origin in vertebrate DCs.

Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel's γ-subunit.

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, ß-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αßγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C-Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.

Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System.

BACKGROUND: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. RESULTS: Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. CONCLUSIONS: Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

The initiation and maintenance of a differentiated state in development.

Proper function of the body is maintained by an intricate interaction and communication among cells. during the animal development how these cells are formed and maintained is an important yet elusive. Understanding of how cells such as muscle and nerve cells maintain their identities would enable us to control the diseases which include malfunctioning in cellular identities such as cancer. In this article, we describe how the concept of formation and maintenance of cell identities has changed over the last 100 years. We will also briefly describe our current experimental work which includes transcriptional dynamics, and protein-protein interaction and how they are bringing new molecular insights. We also describe liquid-liquid phase separation as a potential new mechanism for the stability of gene expression in the non dvididng specialised cells of Xenopus oocytes.

Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

Studying the DNA damage response in embryonic systems.

Maintenance and surveillance of genome integrity is crucial during the very early steps of embryonic development, since de novo mutations generated during this stage can be propagated in differentiated adult cells and may lead to predisposition to diseases including cancer. Surprisingly, early embryos are characterized by a relaxed control of genome integrity, reminiscent of that observed in cancer cells. How embryos manage to produce healthy adult individuals in such conditions remains still unclear. Here, we describe protocols and methods to study and analyze the DNA damage response and genome integrity in two embryonic experimental systems, early Xenopus laevis embryos and mouse embryonic stem cells. We describe methods to study gene functions in the DNA damage response by mRNA microinjection in Xenopus embryos generated by in vitro fertilization, mutagenesis and developmental regulation of the DNA damage response. We also describe methods to analyze the DNA damage response in mESCs, including synchronization experiments that allow studying the DNA damage response at different cell cycle stages. Analysis of genome integrity in these systems may also help to shed light on the molecular mechanisms that preserve genome integrity and become dysregulated in cancer cells.

Meiosis initiation: a story of two sexes in all creatures great and small.

Meiosis facilitates diversity across individuals and serves as a major driver of evolution. However, understanding how meiosis begins is complicated by fundamental differences that exist between sexes and species. Fundamental meiotic research is further hampered by a current lack of human meiotic cells lines. Consequently, much of what we know relies on data from model organisms. However, contextualising findings from yeast, worms, flies and mice can be challenging, due to marked differences in both nomenclature and the relative timing of meiosis. In this review, we set out to combine current knowledge of signalling and transcriptional pathways that control meiosis initiation across the sexes in a variety of organisms. Furthermore, we highlight the emerging links between meiosis initiation and oncogenesis, which might explain the frequent re-expression of normally silent meiotic genes in a variety of human cancers.