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Cell Reports Jun 2022In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the...
In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.
Topics: Blastocyst; Embryonic Development; Gene Expression Regulation, Developmental; Genome; Humans; Zygote
PubMed: 35732112
DOI: 10.1016/j.celrep.2022.110994 -
The EMBO Journal Nov 2022Epigenome reprogramming after fertilization enables transcriptionally quiescent maternal and paternal chromatin to acquire a permissive state for subsequent zygotic...
Epigenome reprogramming after fertilization enables transcriptionally quiescent maternal and paternal chromatin to acquire a permissive state for subsequent zygotic genome activation (ZGA). H3K27 acetylation (H3K27ac) is a well-established chromatin marker of active enhancers and promoters. However, reprogramming dynamics of H3K27ac during maternal-to-zygotic transition (MZT) in mammalian embryos are not well-studied. By profiling the allelic landscape of H3K27ac during mouse MZT, we show that H3K27ac undergoes three waves of rapid global transitions between oocyte stage and 2-cell stage. Notably, germinal vesicle oocyte and zygote chromatin are globally hyperacetylated, with noncanonical, broad H3K27ac domains that correlate with broad H3K4 trimethylation (H3K4me3) and open chromatin. H3K27ac marks genomic regions primed for activation including ZGA genes, retrotransposons, and active alleles of imprinted genes. We show that CBP/p300 and HDAC activities play important roles in regulating H3K27ac dynamics and are essential for preimplantation development. Specifically, CBP/p300 acetyltransferase broadly deposits H3K27ac in zygotes to induce the opening of condensed chromatin at putative enhancers and ensure proper ZGA. On the contrary, HDACs revert broad H3K27ac domains to canonical domains and safeguard ZGA by preventing premature expression of developmental genes. In conclusion, coordinated activities of CBP/p300 and HDACs during mouse MZT are essential for ZGA and preimplantation development.
Topics: Mice; Animals; Zygote; Acetylation; Gene Expression Regulation, Developmental; Blastocyst; Chromatin; Mammals
PubMed: 36215692
DOI: 10.15252/embj.2022112012 -
Genes & Development May 2023Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are...
Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remain unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome. In oocytes, eIF4E1b binds to transcripts encoding translation machinery proteins, chromatin remodelers, and reprogramming factors to promote their translation in zygotes and protect them from degradation. The protein products are thought to establish an open chromatin landscape in one-cell zygotes to enable transcription of genes required for cleavage stage development. Our results define a program for rapid resetting of the zygotic epigenome that is regulated by maternal mRNA expression and provide new insights into the mammalian maternal-to-zygotic transition.
Topics: Animals; Mice; Embryonic Development; Gene Expression Regulation, Developmental; Oocytes; Protein Biosynthesis; RNA, Messenger, Stored; Zygote
PubMed: 37257918
DOI: 10.1101/gad.350400.123 -
Cell Research Dec 2023Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to the inner cell mass (ICM) where pluripotent...
Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to the inner cell mass (ICM) where pluripotent epiblast arises, and extraembryonic trophectoderm. However, how ZGA is connected to the first lineage segregation in mammalian embryos remains elusive. Here, we investigated the role of nuclear receptor (NR) transcription factors (TFs), whose motifs are highly enriched and accessible from the 2-cell (2C) to 8-cell (8C) stages in mouse embryos. We found that NR5A2, an NR TF strongly induced upon ZGA, was required for this connection. Upon Nr5a2 knockdown or knockout, embryos developed beyond 2C normally with the zygotic genome largely activated. However, 4-8C-specific gene activation was substantially impaired and Nr5a2-deficient embryos subsequently arrested at the morula stage. Genome-wide chromatin binding analysis showed that NR5A2-bound cis-regulatory elements in both 2C and 8C embryos are strongly enriched for B1 elements where its binding motif is embedded. NR5A2 was not required for the global opening of its binding sites in 2C embryos but was essential to the opening of its 8C-specific binding sites. These 8C-specific, but not 2C-specific, binding sites are enriched near genes involved in blastocyst and stem cell regulation, and are often bound by master pluripotency TFs in blastocysts and embryonic stem cells (ESCs). Importantly, NR5A2 regulated key pluripotency genes Nanog and Pou5f1/Oct4, and primitive endoderm regulatory genes including Gata6 among many early ICM genes, as well as key trophectoderm regulatory genes including Tead4 and Gata3 at the 8C stage. By contrast, master pluripotency TFs NANOG, SOX2, and OCT4 targeted both early and late ICM genes in mouse ESCs. Taken together, these data identify NR5A2 as a key regulator in totipotent embryos that bridges ZGA to the first lineage segregation during mouse early development.
Topics: Animals; Mice; Blastocyst; Cell Lineage; Embryonic Development; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Transcription Factors; Zygote
PubMed: 37935903
DOI: 10.1038/s41422-023-00887-z -
Epigenetics & Chromatin Dec 2021Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently,...
BACKGROUND
Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated.
RESULTS
We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O), gradient oxygen concentration (5% O to 2% O) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate.
CONCLUSIONS
We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.
Topics: Animals; Blastocyst; Embryo, Mammalian; Embryonic Development; Female; Histones; Mice; Oocytes; Pregnancy; Zygote
PubMed: 34930415
DOI: 10.1186/s13072-021-00431-6 -
Nature Communications Feb 2021Extensive epigenetic reprogramming occurs during preimplantation embryo development. However, it remains largely unclear how the drastic epigenetic reprogramming...
Extensive epigenetic reprogramming occurs during preimplantation embryo development. However, it remains largely unclear how the drastic epigenetic reprogramming contributes to transcriptional regulatory network during this period. Here, we develop a single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell. We apply this method to depict a single-cell multiomics map of mouse preimplantation development. We find that genome-wide DNA methylation remodeling facilitates the reconstruction of genetic lineages in early embryos. Further, we construct a zygotic genome activation (ZGA)-associated regulatory network and reveal coordination among multiple epigenetic layers, transcription factors and repeat elements that instruct proper ZGA. Cell fates associated cis-regulatory elements are activated stepwise in post-ZGA stages. Trophectoderm (TE)-specific transcription factors play dual roles in promoting the TE program while repressing the inner cell mass (ICM) program during the ICM/TE separation.
Topics: Alleles; Animals; Blastocyst; Cell Lineage; Chromatin; DNA Methylation; Ectoderm; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Genomics; Male; Mice; Phylogeny; Promoter Regions, Genetic; Single-Cell Analysis; Zygote
PubMed: 33623021
DOI: 10.1038/s41467-021-21409-8 -
Nature Jun 2023Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise...
Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.
Topics: Animals; Mice; Protein Biosynthesis; Proteomics; Ribosomes; RNA, Messenger; Single-Cell Analysis; Embryonic Development; Alleles; Microfluidic Analytical Techniques; Oocytes; Isotachophoresis; Ribosome Profiling; Centrosome; Zygote
PubMed: 37344592
DOI: 10.1038/s41586-023-06228-9 -
Cell Systems Jul 2020Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex...
Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2, the chromatin remodeler Smarca5, and the transcription factor Patz1, and functional experiments revealed that Smarca5's regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides both system-level and molecular insights into the mechanisms that orchestrate ZGA.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; Epigenesis, Genetic; Genome; Humans; Transcriptome; Zygote
PubMed: 32634384
DOI: 10.1016/j.cels.2020.06.004 -
Nature Communications Oct 2023Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing...
Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.
Topics: Animals; Humans; Transcriptome; Embryonic Development; Zygote; Gene Expression Profiling; Protein Isoforms; Sequence Analysis, RNA; Alternative Splicing; Blastocyst
PubMed: 37903791
DOI: 10.1038/s41467-023-42558-y -
Nature Jan 2024DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme,...
DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome and is considered an epigenetic fingerprint. In spite of its importance in maintaining the epigenome, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.
Topics: Animals; Mice; Blastocyst; Chromatin; DNA Replication Timing; Epigenome; Genome; RNA Polymerase II; Zygote; Embryo, Mammalian
PubMed: 38123678
DOI: 10.1038/s41586-023-06872-1