WGBS and RNA-seq of Danio rerio in HSPC generation
Ontology highlight
ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE189072 | GEO | 2022/05/10
REPOSITORIES: GEO
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Project description:Hematopoietic stem and progenitor cells (HSPCs) are required to establish and maintain the adult blood system in vertebrates. During development, HPSCs are generated from hemogenic endothelial cells that undergo an endothelial-to-hematopoietic transition (EHT). Growth factors and epigenetic changes can promote EHT, but these mechanisms do not explain its tight spatiotemporal regulation during development. Here, we show that microRNA (miR) miR-223-mediated regulation of N-glycan biosynthesis intrinsically restrains EHT, representing a new pathway that prevents excessive HSPC production. We find that miR-223 is uniquely expressed in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR-223 promotes the expansion of these cells in the zebrafish and mouse aorta-gonad-mesonephros (AGM), where EHT occurs. miR-223 targets alg2 (alpha 1,3/ 1,6 mannosyltransferase) in the AGM endothelium to restrict hemogenic and HSPC specification. This enzyme is involved in the attachment of the N-glycans, a common co-translational modification9-12 that influences several pathophysiological processes, but has not yet been implicated in EHT. Using an N-glycosensor, we demonstrate that abundant protein N-glycan attachment occurs in vascular cells during EHT, and this process is required for HSPC production. High-throughput glycome analysis upon loss of miR-223 revealed that terminal alpha 1,3/6 mannose modifications are increased at the expense of alpha 2,3/6 sialic acid sugars. Importantly, pharmacological manipulation targeting these N-glycan types in wild-type embryos phenocopies the loss of miR-223 and enhances EHT as well as HSPC production. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants to restrict the hematopoietic fate.
2021-09-09 | GSE135246 | GEO
Project description:Unlike that of mammals, the total DNA methylome of many cold-blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish, this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just after fertilization for oocyte, as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles, the zygotic mutant zebrafish of dnmt1, the major DNA methylation maintenance methyltransferase, surprisingly can develop to term. To solve the role of DNA methylation in early development, we generated zebrafish embryos derived from dnmt1-knocking down oocytes using a recently developed method OMIS (Oocyte Microinjection in situ), which successfully eliminated DNA methylation before zygotic genome activation. dnmt1-deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53-regulated apoptosis, likely triggered by the derepression of transposable elements. Single cell RNA-seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly, the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers, which acquire ectopic H3K27ac, accessible chromatin, and H3K4me3. These somatic enhancers are preferentially CG-rich and are bound by CG-containing TFs. By contrast, embryonic enhancers are generally CG-poor, methylation-insensitive, and are bound by CG-less TFs. Hence, the global DNA methylome inheritance is essential for vertebrate early development, and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs.
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Project description:Stable inheritance of DNA methylation is critical for maintaining the differentiated phenotypes in multicellular organisms. However, the molecular basis ensuring high fidelity of maintenance DNA methylation is largely unknown. Here, we demonstrate that two distinct modes of DNMT1 recruitment, one is DNA replication-coupled and the other is uncoupled mechanism, regulate the stable inheritance of DNA methylation. PCNA-associated factor 15 (PAF15) represents a primary target of UHRF1 and undergoes dual mono-ubiquitylation (PAF15Ub2) on chromatin. PAF15Ub2 specifically interacts with DNMT1 and controls the recruitment of DNMT1 in a DNA replication-coupled manner. Thus, loss of PAF15Ub2 results in impaired DNA methylation at sites replicating during early S phase. In contrast, outside of S phase or when PAF15 ubiquitylation is perturbed, UHRF1 ubiquitylates histone H3 to promote DNMT1 recruitment. Together, we identify replication-coupled and uncoupled mechanisms of maintenance DNA methylation, both of which collaboratively ensure the stable DNA methylation.
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Project description:Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidence of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA-adenine methylase identification (DamID) method, we identified Dnmt1-binding regions in 4-cell and 8-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refute the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the long interspersed nuclear elements (LINE1) and endogenous retrovirus type-K (ERVK) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding-domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.
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Project description:Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidence of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA-adenine methylase identification (DamID) method, we identified Dnmt1-binding regions in 4-cell and 8-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refute the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the long interspersed nuclear elements (LINE1) and endogenous retrovirus type-K (ERVK) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding-domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.
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Project description:Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidence of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA-adenine methylase identification (DamID) method, we identified Dnmt1-binding regions in 4-cell and 8-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refute the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the long interspersed nuclear elements (LINE1) and endogenous retrovirus type-K (ERVK) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding-domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.
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Project description:Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidence of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA-adenine methylase identification (DamID) method, we identified Dnmt1-binding regions in 4-cell and 8-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refute the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the long interspersed nuclear elements (LINE1) and endogenous retrovirus type-K (ERVK) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding-domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.
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Project description:Zebrafish embryos are transcriptional silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA). We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3’ untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3’ terminal exon when transcribed zygotically relative to the maternal transcript counterparts. ChIP-seq data mapped onto skipped exon events reveals a correlation between histone H3K36trimethylation peaks and the skipped exons, suggesting epigenetic marks being part of alternative splicing regulation. The novel isoforms and isoform switches reported here include regulators of the transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes. Examination H3K36me3 in zebrafish whole embryos at the Post-MBT stage
2012-11-01 | E-GEOD-40629 | biostudies-arrayexpress