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Nature Cell Biology Apr 2024The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if...
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.
Topics: Animals; Chromatin; Gene Expression; Positive Transcriptional Elongation Factor B; RNA Polymerase II; Transcription, Genetic; Zebrafish
PubMed: 38589534
DOI: 10.1038/s41556-024-01389-9 -
Genetics Jun 2024Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce...
Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon "chromoanasynthesis." These data show that Recql5 mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs.
Topics: Animals; RecQ Helicases; Mice; CRISPR-Cas Systems; Zygote; Mutation
PubMed: 38577877
DOI: 10.1093/genetics/iyae054 -
Nucleic Acids Research Jun 2024In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic...
In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologically associating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage.
Topics: Animals; Mice; Histones; Gene Expression Regulation, Developmental; Chromatin; Transcription, Genetic; Zygote; Gene Knockdown Techniques; Female
PubMed: 38567720
DOI: 10.1093/nar/gkae214 -
Genome Biology Apr 2024Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However,...
BACKGROUND
Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure.
RESULTS
Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization.
CONCLUSION
Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.
Topics: DNA Methylation; Oryza; Seeds; Methyltransferases; Gametogenesis; Gene Expression Regulation, Plant
PubMed: 38566207
DOI: 10.1186/s13059-024-03222-w -
Animal Reproduction 2024The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the...
The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of transcripts in somatic tissues. Notably, the expression levels of in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo.
PubMed: 38562607
DOI: 10.1590/1984-3143-AR2023-0146 -
Animal Reproduction 2024One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of...
One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.
PubMed: 38562606
DOI: 10.1590/1984-3143-AR2023-0107 -
Molecular Therapy. Nucleic Acids Jun 2024Efficient germline mtDNA editing is required to construct disease-related animal models and future gene therapy. Recently, the DddA-derived cytosine base editors...
Efficient germline mtDNA editing is required to construct disease-related animal models and future gene therapy. Recently, the DddA-derived cytosine base editors (DdCBEs) have made mitochondrial genome (mtDNA) precise editing possible. However, there still exist challenges for editing some mtDNA sites in germline via zygote injection, probably due to the suspended mtDNA replication during preimplantation development. Here, we introduce a germline mtDNA base editing strategy: injecting DdCBEs into oocytes of secondary follicles, at which stage mtDNA replicates actively. With this method, we successfully observed efficient G-to-A conversion at a hard-to-edit site and also obtained live animal models. In addition, for those editable sites, this strategy can greatly improve the base editing efficiency up to 3-fold, which is more than that in zygotes. More important, editing in secondary follicles did not increase more the risk of off-target effects than that in zygotes. This strategy provides an option to efficiently manipulate mtDNA sites in germline, especially for hard-to-edit sites.
PubMed: 38560422
DOI: 10.1016/j.omtn.2024.102170 -
Frontiers in Cell and Developmental... 2024Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced...
Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated . The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both and , using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. This method circumvents the intricate procedures involved in embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.
PubMed: 38559814
DOI: 10.3389/fcell.2024.1376936 -
Frontiers in Cell and Developmental... 2024A conserved process of early embryonic development in metazoans is the reductive cell divisions following oocyte fertilization, termed cell cleavages. Cell cleavage... (Review)
Review
A conserved process of early embryonic development in metazoans is the reductive cell divisions following oocyte fertilization, termed cell cleavages. Cell cleavage cycles usually start synchronously, lengthen differentially between the embryonic cells becoming asynchronous, and cease before major morphogenetic events, such as germ layer formation and gastrulation. Despite exhibiting species-specific characteristics, the regulation of cell cleavage dynamics comes down to common controllers acting mostly at the single cell/nucleus level, such as nucleus-to-cytoplasmic ratio and zygotic genome activation. Remarkably, recent work has linked cell cleavage dynamics to the emergence of collective behavior during embryogenesis, including pattern formation and changes in embryo-scale mechanics, raising the question how single-cell controllers coordinate embryo-scale processes. In this review, we summarize studies across species where an association between cell cleavages and collective behavior was made, discuss the underlying mechanisms, and propose that cell-to-cell variability in cell cleavage dynamics can serve as a mechanism of long-range coordination in developing embryos.
PubMed: 38559810
DOI: 10.3389/fcell.2024.1358971 -
BioRxiv : the Preprint Server For... Jun 2024Fertilization occurs before completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within zygotes of mouse and . If...
Fertilization occurs before completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within zygotes of mouse and . If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in , we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.
PubMed: 38559153
DOI: 10.1101/2024.03.12.584242