59 Genome activation in intracytoplasmic sperm injection-derived horse embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 155 ◽  
Author(s):  
D. E. Goszczynski ◽  
P. Tinetti ◽  
Y. H. Choi ◽  
K. Hinrichs ◽  
P. J. Ross
Keyword(s):  
Gene Ontology ◽  
Intracytoplasmic Sperm Injection ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
Cell Stage ◽  
Paternal Genome ◽  
Informative Snps ◽  
Upregulated Genes ◽  
Genome Activation ◽  
Cell Transition

During pre-implantation development, embryos go through a critical period of embryonic genome activation (EGA). The timing of EGA is species specific, but little is known in horse embryos. Here, we aimed to characterise EGA in equine embryos produced by intracytoplasmic sperm injection. Embryos were produced by intracytoplasmic sperm injection of oocytes from 3 mares. Two embryos from each mare at each of 8 developmental stages (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) were individually analysed by RNA-seq. Differential expression was evaluated using binomial Wald tests with an absolute logFC (fold change) threshold of 1 in the DESEqn 2R package. We found that EGA occurred in a two-step fashion. Minor EGA took place during the 2-cell to 4-cell transition, and featured up-regulation of 751 genes and discrete down-regulation of 60 genes in 4-cell embryos compared with 2-cell embryos. Differentially upregulated genes were enriched in gene ontology terms related to transcriptional activator activity, homeobox domains, and nucleosome assembly. Major EGA occurred during the 4-cell to 8-cell transition and included the largest number of differentially expressed genes (n=2,023) between consecutive stages. This period also featured the first massive transcript downregulation (n=816). Upregulated genes were enriched in gene ontology terms related to ribosomal assembly, translation, and RNA modification. Additionally, we observed that the number of intronic sequences was significantly higher from the 4-cell stage onward, indicating active transcription in comparison to oocytes, zygotes, and 2-cell embryos. To evaluate the timing of paternal genome activation, we used whole-genome sequencing data from the parents (average genome coverage of 19×) to quantify allele-specific expression. The average number of informative SNPs in exons, i.e. SNPs with alternative homozygous genotypes from the sire (AA mare - BB sire), was 26 128 per mare, corresponding to 7696 genes. Parental-specific transcript abundance was determined for each embryo, with an average of 1,911±865 informative SNPs detected per sample. Paternal alleles were considered expressed when they reached 10% of the maternal count. Across development, paternal transcripts became appreciable at the 4-cell stage, with 14.15±7.60% of the informative SNPs exhibiting paternal expression, and increased thereafter until reaching a maximum of 96.34% at the blastocyst stage. Overall, this work demonstrates that EGA in horse embryos starts at the 4-cell stage and achieves its main activation at the 8-cell stage. Further analysis will be performed to detail paternal vs. maternal gene expression at the different embryonic stages.

scholarly journals Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julien Richard Albert ◽  
Wan Kin Au Yeung ◽  
Keisuke Toriyama ◽  
Hisato Kobayashi ◽  
Ryutaro Hirasawa ◽  
...  
Keyword(s):  
De Novo ◽  
Cpg Islands ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mass Spectrometry Analysis ◽  
Paternal Allele ◽  
Sequencing Data ◽  
Cell Stage ◽  
Paternal Genome ◽  
Spectrometry Analysis

Abstract De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

scholarly journals Genome reprogramming during the first cell cycle in in vitro produced porcine embryos

2010 ◽  
Vol 55 (No. 2) ◽  
pp. 49-57 ◽  
Author(s):  
I. Barnetová ◽  
K. Okada
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Methylation Pattern ◽  
Dna Demethylation ◽  
Embryo Production ◽  
Cell Stage ◽  
Paternal Genome ◽  
Labelling Intensity ◽  
Mammalian Embryos ◽  
Methylation Patterns

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in <i>in vitro</i> produced porcine zygotes, whereas <i>in vitro</i> produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two <i>in vitro</i> techniques commonly used for <i>in vitro</i > embryo production (<i>in vitro</i> fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after <i>in vitro</i> fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that <i>in vitro</i> matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the <i>in vitro</i> technique for embryo production.

scholarly journals Whole-genome analysis of noncoding genetic variations identifies multigranular regulatory element perturbations associated with Hirschsprung disease

2020 ◽  
Author(s):  
Alexander Xi Fu ◽  
Kathy Nga-Chu Lui ◽  
Clara Sze-Man Tang ◽  
Ray Kit Ng ◽  
Frank Pui-Ling Lai ◽  
...  
Keyword(s):  
Neural Crest ◽  
Genetic Variants ◽  
Regulatory Element ◽  
Hirschsprung Disease ◽  
Regulatory Elements ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Sequencing Data ◽  
Whole Genome Analysis ◽  
Cell Stage

AbstractIt is widely recognized that the missing heritability of many human diseases is partially due to noncoding genetic variants, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals, and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multi-granular information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell-stage-specific regulatory roles three top novel regulatory elements on our list, respectively in the RET, RASGEF1A and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the affects the binding of the gliogenesis regulator NFIA, with a corresponding down-regulation of multiple genes in the same topologically associating domain.

scholarly journals Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kohtaro Morita ◽  
Yuki Hatanaka ◽  
Shunya Ihashi ◽  
Masahide Asano ◽  
Kei Miyamoto ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Arginine Methylation ◽  
Dna Demethylation ◽  
Zygotic Genome Activation ◽  
H3k4 Methylation ◽  
Cell Stage ◽  
Paternal Genome ◽  
Genome Activation ◽  
Histone Arginine Methylation ◽  
Zygotic Genome

AbstractPaternal genome reprogramming, such as protamine–histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

scholarly journals Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

2021 ◽  
Author(s):  
Eric S Tvedte ◽  
Mark Gasser ◽  
Benjamin C Sparklin ◽  
Jane Michalski ◽  
Carl E Hjelmen ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Hybrid Approach ◽  
Cost Effective ◽  
Fruit Fly ◽  
Drosophila Ananassae ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
E Coli ◽  
Hybrid Approaches ◽  
Long Read

Abstract The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

scholarly journals A 127 kb truncating deletion of PGRMC1 is a novel cause of X-linked isolated paediatric cataract

2021 ◽  
Author(s):  
Johanna L. Jones ◽  
Mark A. Corbett ◽  
Elise Yeaman ◽  
Duran Zhao ◽  
Jozef Gecz ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Protein Interactions ◽  
Cholesterol Biosynthesis ◽  
Crystalline Lens ◽  
Whole Genome Sequencing Data ◽  
Mendelian Disease ◽  
Cataract Formation ◽  
Sequencing Data ◽  
Zebrafish Model ◽  
Paediatric Cataract

AbstractInherited paediatric cataract is a rare Mendelian disease that results in visual impairment or blindness due to a clouding of the eye’s crystalline lens. Here we report an Australian family with isolated paediatric cataract, which we had previously mapped to Xq24. Linkage at Xq24–25 (LOD = 2.53) was confirmed, and the region refined with a denser marker map. In addition, two autosomal regions with suggestive evidence of linkage were observed. A segregating 127 kb deletion (chrX:g.118373226_118500408del) in the Xq24–25 linkage region was identified from whole-genome sequencing data. This deletion completely removed a commonly deleted long non-coding RNA gene LOC101928336 and truncated the protein coding progesterone receptor membrane component 1 (PGRMC1) gene following exon 1. A literature search revealed a report of two unrelated males with non-syndromic intellectual disability, as well as congenital cataract, who had contiguous gene deletions that accounted for their intellectual disability but also disrupted the PGRMC1 gene. A morpholino-induced pgrmc1 knockdown in a zebrafish model produced significant cataract formation, supporting a role for PGRMC1 in lens development and cataract formation. We hypothesise that the loss of PGRMC1 causes cataract through disrupted PGRMC1-CYP51A1 protein–protein interactions and altered cholesterol biosynthesis. The cause of paediatric cataract in this family is the truncating deletion of PGRMC1, which we report as a novel cataract gene.

scholarly journals Human-lineage-specific genomic elements are associated with neurodegenerative disease and APOE transcript usage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhongbo Chen ◽  
◽  
David Zhang ◽  
Regina H. Reynolds ◽  
Emil K. Gustavsson ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Purifying Selection ◽  
Whole Genome Sequencing Data ◽  
Human Lineage ◽  
Sequencing Data ◽  
Protein Coding ◽  
Potential Association ◽  
High Depth ◽  
Specific Sequences ◽  
Human Specific

AbstractKnowledge of genomic features specific to the human lineage may provide insights into brain-related diseases. We leverage high-depth whole genome sequencing data to generate a combined annotation identifying regions simultaneously depleted for genetic variation (constrained regions) and poorly conserved across primates. We propose that these constrained, non-conserved regions (CNCRs) have been subject to human-specific purifying selection and are enriched for brain-specific elements. We find that CNCRs are depleted from protein-coding genes but enriched within lncRNAs. We demonstrate that per-SNP heritability of a range of brain-relevant phenotypes are enriched within CNCRs. We find that genes implicated in neurological diseases have high CNCR density, including APOE, highlighting an unannotated intron-3 retention event. Using human brain RNA-sequencing data, we show the intron-3-retaining transcript to be more abundant in Alzheimer’s disease with more severe tau and amyloid pathological burden. Thus, we demonstrate potential association of human-lineage-specific sequences in brain development and neurological disease.

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