Zygotic Gene Activation Research Articles

Single-cell analysis of preimplantation embryonic development in guinea pigs

BackgroundGuinea pigs exhibit numerous physiological similarities to humans, yet the details of their preimplantation embryonic development remain largely unexplored.ResultsTo address this, we conducted single-cell sequencing on the transcriptomes of cells isolated from the zygote stage through preimplantation stages in guinea pigs. This study identified seven distinct cell types within guinea pig preimplantation embryos and pinpointed the timing of zygotic gene activation (ZGA). Trajectory analysis revealed a bifurcation into two lineage-specific branches, accompanied by alterations in specific pathways, including oxidative phosphorylation and vascular endothelial growth factor (VEGF). Additionally, co-expressed gene network analysis highlighted the most enriched functional modules for the epiblast (EPI), primitive endoderm (PrE), and inner cell mass (ICM). Finally, we compared the similarities and differences between human and guinea pig epiblasts (EPIs).ConclusionThis study systematically constructs a cell atlas of guinea pig preimplantation embryonic development, offering fresh insights into mammalian embryonic development and providing alternative experimental models for studying human embryonic development.

Heat tolerance, oxidative stress response tuning and robust gene activation in early-stage Drosophila melanogaster embryos.

In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos differed in both constitutive and heat-stress-induced responses of the expression of relatively few genes, including genes involved in oxidative stress. Most of the transcriptomic response to heat stress was shared among all embryos. Embryos shifted the expression of thousands of genes, including increases in the expression of heat shock genes, suggesting robust zygotic gene activation and demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and developmental genetics.

Lactate modulates zygotic genome activation through H3K18 lactylation rather than H3K27 acetylation

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications.

P-232 EIF4E1B Contributes to Early Translation Burst for Embryogenesis Whose Deficiency Causes Embryo Development Arrest at Four-Cell Stage during Oocyte to Embryo Transition in Women

Abstract Study question What are the effects and corresponding mechanisms of the early burst of translation inhibited during the oocyte-to-embryo transition (OET), which is essential for human embryogenesis? Summary answer EIF4E1B contributes to the early burst of translation for embryogenesis whose deficiency causes embryo development arrest at four-cell stage during OET in women. What is known already The first records of human zygotic genes are observed in late one-cell zygotes, and translation in mammals happens before the activation of zygotic genes. Recently, we identified an oocyte specific role of eIF4E1b in selecting and regulating the translation initiation of individual mRNAs that are involved in cell differentiation, cell cycle regulation, and cell fate determination during OET. The genetic ablation of Eif4e1b in mice resulted in a developmental arrest in the 2-cell phase of embryogenesis. How is the early burst of translation regulated in human embryogenesis remains unknown. Study design, size, duration A total of 236 female Chinese participants who underwent assisted reproductive technology (ART) and experienced at least one cycle of OET defects were recruited from our university-affiliated center from January 2017 to May 2023. 60 frozen human 3 pronuclei (3PN) embryos have been used for this work. The use of human embryos donated for research was allowed by the the Ethics Committee of the Reproductive and Genetic Hospital of CITIC–Xiangya (reference LL-SC-2018-009). Participants/materials, setting, methods All recruited individuals underwent whole-exome sequencing. In vitro transcription and micro-injection were employed to validate the embryo development pattern under different translation inhibition as well as potential effects of the EIF4E1B variant in mouse oocytes. Immunoprecipitation was conducted to investigate the causes of OET failure and identify the ability of eIF4E1B bind with PABPC1L. Additionally, a knock-in (KI) mouse model carrying homozygous Eif4e1bA192T was generated to evaluate female fertility and a more comprehensive mechanism. Main results and the role of chance Translation inhibitor cycloheximide (CHX) and knockdown of different translation initiation factors eIF4Es were administered to human 3 pronuclei (3PN) embryos, starting from the 1C stage. The absence of eIF4E1B was identified as a specific phenotype, leading to developmental arrest at the 1-4C stage, which is consistent with the effects of Translation inhibitor CHX. Additionally, a novel homozygous mutation (c.565G>A; p. Ala189Thr) in EIF4E1B was found within a consanguineous family. This mutation was associated with recurrent four-cell arrests, suggesting a translational regulation role for eIF4E1B from the 1C to 4C stage during OET. The mutated eIF4E1B disrupted the expression and condensate distribution of eIF4E1B. We also confirmed the interaction between eIF4E1B and PABPC1L, with the c.565G>A variant in EIF4E1B impairing their binding ability. Furthermore, KI mice carrying homozygous Eif4e1bA192T partially recapitulated the phenotype resulting from EIF4E1B deficiency. Limitations, reasons for caution More cases are required to demonstrate the association between EIF4E1B variants and phenotypes. Wider implications of the findings By delving deeper into the intricate interactions between basic physiology and clinical characteristics, this research elucidates the crucial role of maternal EIF4E1B in ensuring the early burst of translation for embryogenesis and provide a diagnostic genetic marker for women with OET defects. Trial registration number Not applicable

RNA helicase DEAD-box-5 is involved in R-loop dynamics of preimplantation embryos.

R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

The impact of selective HDAC inhibitors on the transcriptome of early mouse embryos

BackgroundHistone acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a crucial role in the control of gene expression. HDAC inhibitors (HDACi) have shown potential in cancer therapy; however, the specific roles of HDACs in early embryos remain unclear. Moreover, although some pan-HDACi have been used to maintain cellular undifferentiated states in early embryos, the specific mechanisms underlying their effects remain unknown. Thus, there remains a significant knowledge gap regarding the application of selective HDACi in early embryos.ResultsTo address this gap, we treated early embryos with two selective HDACi (MGCD0103 and T247). Subsequently, we collected and analyzed their transcriptome data at different developmental stages. Our findings unveiled a significant effect of HDACi treatment during the crucial 2-cell stage of zygotes, leading to a delay in embryonic development after T247 and an arrest at 2-cell stage after MGCD0103 administration. Furthermore, we elucidated the regulatory targets underlying this arrested embryonic development, which pinpointed the G2/M phase as the potential period of embryonic development arrest caused by MGCD0103. Moreover, our investigation provided a comprehensive profile of the biological processes that are affected by HDACi, with their main effects being predominantly localized in four aspects of zygotic gene activation (ZGA): RNA splicing, cell cycle regulation, autophagy, and transcription factor regulation. By exploring the transcriptional regulation and epigenetic features of the genes affected by HDACi, we made inferences regarding the potential main pathways via which HDACs affect gene expression in early embryos. Notably, Hdac7 exhibited a distinct response, highlighting its potential as a key player in early embryonic development.ConclusionsOur study conducted a comprehensive analysis of the effects of HDACi on early embryonic development at the transcriptional level. The results demonstrated that HDACi significantly affected ZGA in embryos, elucidated the distinct actions of various selective HDACi, and identified specific biological pathways and mechanisms via which these inhibitors modulated early embryonic development.

DUX4 expression in cancer induces a metastable early embryonic totipotent program

The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the DUX4-regulated ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages, while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions increased the fraction of cells expressing DUX4 and its downstream programs. Our demonstration that transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and suppresses antigen presentation has implications for cancer growth, progression, and immune evasion.

O-058 Metabolism, histone modification and early embryo development

Abstract Cellular metabolism provides fundamental components for chromatin dynamics and epigenetics. However, metabolic features, and their impact on epigenetic remodeling during mammalian pre-implantation development, remain poorly understood. In this study, we established the metabolic landscape of mouse pre-implantation embryos from zygote to blastocyst, and quantified their absolute carbohydrate metabolites. We found that amino acid/carbohydrate/lipid metabolism increased during pre-implantation development, whereas nucleotide metabolism increased at the 4- and 8-cell stages. We further demonstrated that oxidized nicotinamide adenine dinucleotide (NAD+) is indispensable for mouse pre-implantation development. Mechanistically, NAD+ avoids excessive minor zygotic gene activation (ZGA) by cooperating with deacylase SIRT1 to remove zygotic H3K27ac. In human, NAD+ supplement can promote the removal of zygotic H3K27ac and benefit preimplantation development. Our findings demonstrate that precise and timely regulation of minor ZGA is controlled by metabolic dynamics, and enhance our understanding of the metabolism of mammalian early embryos.

Transcription factor ELK1 regulates the expression of histone 3 lysine 9 to affect developmental potential of porcine preimplantation embryos

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

Cell-specific effects of the sole C. elegans Daughterless/E protein homolog, HLH-2, on nervous system development.

Are there common mechanisms of neurogenesis used throughout an entire nervous system? We explored to what extent canonical proneural class I/II bHLH complexes are responsible for neurogenesis throughout the entire Caenorhabditis elegans nervous system. Distinct, lineage-specific proneural class II bHLH factors are generally thought to operate via interaction with a common, class I bHLH subunit, encoded by Daughterless in flies, the E proteins in vertebrates and HLH-2 in C. elegans. To eliminate function of all proneuronal class I/II bHLH complexes, we therefore genetically removed maternal and zygotic hlh-2 gene activity. We observed broad effects on neurogenesis, but still detected normal neurogenesis in many distinct neuron-producing lineages of the central and peripheral nervous system. Moreover, we found that hlh-2 selectively affects some aspects of neuron differentiation while leaving others unaffected. Although our studies confirm the function of proneuronal class I/II bHLH complexes in many different lineages throughout a nervous system, we conclude that their function is not universal, but rather restricted by lineage, cell type and components of differentiation programs affected.

Nudt7 gene depletion causes transcriptomic change in early development of zebrafish.

The Nudt family has been identified as enzymes performing Coenzyme A to 3'5'-ADP + 4'-phospho pantetheine catalysis. The members of this family have been shown to be particularly involved in lipid metabolism, while their involvement in gene regulation through regulating transcription or mRNA metabolism has also been suggested. Here, we focused on peroxisomal NUDT7, possessing enzymatic activity similar to that of its paralog, peroxisomal NUDT19, which is involved in mRNA degradation. No reports have been published about the Nudt family in zebrafish. Our transcriptomic data showed that the Nudt family members are highly expressed around zygotic gene activation (ZGA) in developing zebrafish embryos. Therefore, we confirmed the computational prediction that the products of the nudt7 gene in zebrafish were localized in the peroxisome and highly expressed in early embryogenesis. The depletion of nudt7 genes by the CRISPR/Cas9 system did not affect development; however, it decreased the rate of transcription in ZGA. In addition, H3K27ac ChIP-seq analysis demonstrated that this decrease in transcription was correlated with the genome-wide decrease of H3K27ac level. This study suggests that peroxisomal Nudt7 functions in regulating transcription in ZGA via formation of the H3K27ac domain in active chromatin.

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

BackgroundThe process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially contribute to dissecting early embryonic processes.ResultsWe present genome-wide identification of candidate genes that exhibit locally restricted expression in germ disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi.ConclusionsOur genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

Metabolic control of histone acetylation for precise and timely regulation of minor ZGA in early mammalian embryos

Metabolism feeds into the regulation of epigenetics via metabolic enzymes and metabolites. However, metabolic features, and their impact on epigenetic remodeling during mammalian pre-implantation development, remain poorly understood. In this study, we established the metabolic landscape of mouse pre-implantation embryos from zygote to blastocyst, and quantified some absolute carbohydrate metabolites. We integrated these data with transcriptomic and proteomic data, and discovered the metabolic characteristics of the development process, including the activation of methionine cycle from 8-cell embryo to blastocyst, high glutaminolysis metabolism at blastocyst stage, enhanced TCA cycle activity from the 8-cell embryo stage, and active glycolysis in the blastocyst. We further demonstrated that oxidized nicotinamide adenine dinucleotide (NAD+) synthesis is indispensable for mouse pre-implantation development. Mechanistically, in part, NAD+ is required for the exit of minor zygotic gene activation (ZGA) by cooperating with SIRT1 to remove zygotic H3K27ac. In human, NAD+ supplement can promote the removal of zygotic H3K27ac and benefit pre-implantation development. Our findings demonstrate that precise and timely regulation of minor ZGA is controlled by metabolic dynamics, and enhance our understanding of the metabolism of mammalian early embryos.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

USP16-mediated histone H2A lysine-119 deubiquitination during oocyte maturation is a prerequisite for zygotic genome activation.

Maternal-to-zygotic transition (MZT) is the first and key step in the control of animal development and intimately related to changes in chromatin structure and histone modifications. H2AK119ub1, an important epigenetic modification in regulating chromatin configuration and function, is primarily catalyzed by PRC1 and contributes to resistance to transcriptional reprogramming in mouse embryos. In this study, the genome-wide dynamic distribution of H2AK119ub1 during MZT in mice was investigated using chromosome immunoprecipitation and sequencing. The results indicated that H2AK119ub1 accumulated in fully grown oocytes and was enriched at the TSSs of maternal genes, but was promptly declined after meiotic resumption at genome-wide including the TSSs of early zygotic genes, by a previously unidentified mechanism. Genetic evidences indicated that ubiquitin-specific peptidase 16 (USP16) is the major deubiquitinase for H2AK119ub1 in mouse oocytes. Conditional knockout of Usp16 in oocytes did not impair their survival, growth, or meiotic maturation. However, oocytes lacking USP16 have defects when undergoing zygotic genome activation or gaining developmental competence after fertilization, potentially associated with high levels of maternal H2AK119ub1 deposition on the zygotic genomes. Taken together, H2AK119ub1 level is declined during oocyte maturation by an USP16-dependent mechanism, which ensures zygotic genome reprogramming and transcriptional activation of essential early zygotic genes.

Zygotic gene activation in mice: profile and regulation.

The zygotic genome is transcriptionally silent immediately after fertilization. In mice, initial activation of the zygotic genome occurs in the middle of the one-cell stage. At the mid-to-late two-cell stage, a burst of gene activation occurs after the second round of DNA replication, and the profile of transcribed genes changes dramatically. These two phases of gene activation are called minor and major zygotic gene activation (ZGA), respectively. As they mark the beginning of the gene expression program, it is important to elucidate gene expression regulation during these stages. This article reviews the outcomes of studies that have clarified the profiles and regulatory mechanisms of ZGA.

Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

The endogenous mex-3 3´UTR is required for germline repression and contributes to optimal fecundity in C. elegans.

RNA regulation is essential to successful reproduction. Messenger RNAs delivered from parent to progeny govern early embryonic development. RNA-binding proteins (RBPs) are the key effectors of this process, regulating the translation and stability of parental transcripts to control cell fate specification events prior to zygotic gene activation. The KH-domain RBP MEX-3 is conserved from nematode to human. It was first discovered in Caenorhabditis elegans, where it is essential for anterior cell fate and embryo viability. Here, we show that loss of the endogenous mex-3 3´UTR disrupts its germline expression pattern. An allelic series of 3´UTR deletion variants identify repressing regions of the UTR and demonstrate that repression is not precisely coupled to reproductive success. We also show that several RBPs regulate mex-3 mRNA through its 3´UTR to define its unique germline spatiotemporal expression pattern. Additionally, we find that both poly(A) tail length control and the translation initiation factor IFE-3 contribute to its expression pattern. Together, our results establish the importance of the mex-3 3´UTR to reproductive health and its expression in the germline. Our results suggest that additional mechanisms control MEX-3 function when 3´UTR regulation is compromised.

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) dietalters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.