The epigenome is dynamic and forged by epigenetic mechanisms, such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA species. Increasing lines of evidence support the concept that certain acquired traits are derived from environmental exposure during early embryonic and fetal development, i.e., fetal programming, and can even be "memorized" in the germline as epigenetic information and transmitted to future generations. Advances in technology are now driving the global profiling and precise editing of germline and embryonic epigenomes, thereby improving our understanding of epigenetic regulation and inheritance. These achievements open new avenues for the development of technologies or potential management interventions to counteract adverse conditions or improve performance in livestock species. In this article, we review the epigenetic analyses (DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs) of germ cells and embryos in mammalian livestock species (cattle, sheep, goats, and pigs) and the epigenetic determinants of gamete and embryo viability. We also discuss the effects of parental environmental exposures on the epigenetics of gametes and the early embryo, and evidence for transgenerational inheritance in livestock.

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The Epigenetics of Gametes and Early Embryos and Potential Long-Range Consequences in Livestock Species-Filling in the Picture With Epigenomic Analyses.

Ruminant Genome Database (RGD; http://animal.nwsuaf.edu.cn/RGD) provides visualization and analysis tools for ruminant comparative genomics and functional annotations. As more high-quality ruminant genome assemblies have become available, we have redesigned the user interface, integrated and expanded multi-omics data, and developed novel features to improve the database. The new version, RGD v2.0, houses 78 ruminant genomes; 110-species synteny alignments for major livestock (including cattle, sheep, goat) and wild ungulates; 21 012 orthologous gene clusters with Gene Ontology and pathway annotation; ∼8 600 000 conserved elements; and ∼1 000 000 cis-regulatory elements by utilizing 1053 epigenomic data sets. The transcriptome data in RGD v2.0 has nearly doubled, currently with 1936 RNA-seq data sets, and 155 174 phenotypic data sets have been newly added. New and updated features include: (i) The UCSC Genome Browser, BLAT, BLAST and Table Browser tools were updated for six available ruminant livestock species. (ii) The LiftOver tool was newly introduced into our browser to allow coordinate conversion between different ruminant assemblies. And (iii) tissue specificity index, tau, was calculated to facilitate batch screening of specifically expressed genes. The enhanced genome annotations and improved functionality in RGD v2.0 will be useful for study of genome evolution, environmental adaption, livestock breeding and biomedicine.

RGD v2.0: a major update of the ruminant functional and evolutionary genomics database.

The bovine represents an important agriculture species and dairy breeds have experienced intense genetic selection over the last decades. The selection of breeders focused initially on milk production, but now includes feed efficiency, health, and fertility, although these traits show lower heritability. The non-genetic paternal and maternal effects on the next generation represent a new research topic that is part of epigenetics. The evidence for embryo programming from both parents is increasing. Both oocytes and spermatozoa carry methylation marks, histones modifications, small RNAs, and chromatin state variations. These epigenetic modifications may remain active in the early zygote and influence the embryonic period and beyond. In this paper, we review parental non-genetic effects retained in gametes on early embryo development of dairy cows, with emphasis on parental age (around puberty), the metabolism of the mother at the time of conception and in vitro culture (IVC) conditions. In our recent findings, transcriptomic signatures and DNA methylation patterns of blastocysts and gametes originating from various parental and IVC conditions revealed surprisingly similar results. Embryos from all these experiments displayed a metabolic signature that could be described as an "economy" mode where protein synthesis is reduced, mitochondria are considered less functional. In the absence of any significant phenotype, these results indicated a possible similar adaptation of the embryo to younger parental age, post-partum metabolic status and IVC conditions mediated by epigenetic factors.

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Parental Effects on Epigenetic Programming in Gametes and Embryos of Dairy Cows

The development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms, which depend on chromatin accessibility However, how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development, we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45, 70 and 100 days post coitus (dpc) In total, 21,638, 35,447 and 60,181 unique regions (or peaks) were found across the embryos at 45 dpc (LW45), 70 dpc (LW70) and 100 dpc (LW100), respectively More than 91% of the peaks were annotated within − 1 kb to 100 bp of transcription start sites (TSSs) First, widespread increases in specific accessible chromatin regions (ACRs) from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development Second, the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes Moreover, the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle development-related genes, such as MyoG, Mef2c, and Mef2d Several potential transcriptional repressors, including E2F6, OTX2 and CTCF, were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data This work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development

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The landscape of chromatin accessibility in skeletal muscle during embryonic development in pigs

After myocardial infarction, the massive death of cardiomyocytes leads to cardiac fibroblast proliferation and myofibroblast differentiation, which contributes to the extracellular matrix remodeling of the infarcted myocardium. We recently found that myofibroblasts further differentiate into matrifibrocytes, a newly identified cardiac fibroblast differentiation state. Cardiac fibroblasts of different states have distinct gene expression profiles closely related to their functions. However, the mechanism responsible for the gene expression changes during these activation and differentiation events is still not clear. In this study, the gene expression profiling and genome-wide accessible chromatin mapping of mouse cardiac fibroblasts isolated from the uninjured myocardium and the infarct at multiple time points corresponding to different differentiation states were performed by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. ATAC-seq peaks were highly enriched in the promoter area and the distal area where enhancers are located. A positive correlation was identified between the expression and promoter accessibility for many dynamically expressed genes, even though evidence showed that mechanisms independent of chromatin accessibility may also contribute to the gene expression changes in cardiac fibroblasts after MI. Moreover, motif enrichment analysis and gene regulatory network construction identified transcription factors that possibly contributed to the differential gene expression between cardiac fibroblasts of different states.

The landscape of accessible chromatin in quiescent cardiac fibroblasts and cardiac fibroblasts activated after myocardial infarction.

Chromatin reorganization governs the regulation of gene expression during preimplantation development. However, the landscape of chromatin dynamics in this period has not been explored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) which revealed unique features of the accessible chromatin during bovine early embryo development. We found that chromatin accessibility is low in oocytes and 2-/4-cell embryos, followed by a significant increase in embryos during major embryonic genome activation (EGA), and peaked in elongating day 14 embryos. Genome-wide characteristics of open chromatin showed that ATAC-seq signals in both transcription start sites (TSS) and transcription end sites (TES) were strong. Additionally, the distal ATAC-seq peaks were enriched in repeat elements in a type-specific and stage-specific manner. We further unveiled a series of transcription factor (TF) motifs with distinct variation of enrichment from distal ATAC-seq peaks. By integrated analysis of chromatin accessibility with transcriptomes and DNA methylomes in bovine early embryos, we showed that promoter accessibility was positively correlated with gene expression, especially during major EGA, and was strongly correlated to DNA methylation and CpG density. Finally, we identified the critical chromatin signatures and TFs that differ between in vivo and in vitro derived blastocysts, which provides insights to the potential mechanisms leading to low quality of embryos produced in vitro. Together, this comprehensive analysis revealed critical features of chromatin landscape and epigenetic reprogramming during bovine preimplantation embryo development.

The landscape of accessible chromatin in bovine oocytes and early embryos.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.

The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes.

Chromatin reorganization governs gene expression regulation during pre-implantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We analysed pools of 20 germinal vesicles or MII oocytes or 2-, 4-, 8-, 16-cell, morula, and blastocyst stage in vitro-produced embryos. We conducted ATAC-seq on six pools for each stage and an additional four pools of in vivo-derived morula and blastocysts and six replicates using individual Day 14 elongating embryos. We obtained ~110 million paired end reads uniquely mapped to the bovine reference genome for each stage. Hierarchical clustering, t-distributed stochastic neighbour embedding, and principal component analysis showed four distinct patterns for open chromatin status: (1) low accessibility in germinal vesicles and MII oocytes and in 2- and 4-cell embryos; (2) significantly elevated accessibility in 8-cell, 16-cell, and morula embryos; (3) less accessibility in blastocysts; and (4) extremely high accessibility in elongating embryos. This dynamic and sequential chromatin remodelling is consistent with transcription activation during the bovine minor embryonic genome activation from fertilization to 4-cell, major embryonic genome activation at 8-cell, first differentiation at blastocyst and drastic transcription initiation for embryo elongation. Genome-wide characteristics of accessible chromatin showed (1) accessible chromatin near the transcription start sites of active genes and CpG-rich promoters; (2) widespread accessible chromatin regions extensively overlapped with transposable elements; (3) distal peaks preferentially enriched for repeats including LINE, SINE, and LTR from 8-cell to morula embryos, especially for LTR, whereas enrichment in simple repeats were found from oocytes to 4-cell and in elongating embryos; and (4) highly stage-specific transcription factor motifs in distal peaks were unveiled. By integrating the maps of chromatin accessibility with bovine embryo transcriptomes and DNA methylomes, we found promoter accessibility and DNA methylation in bovine embryos correlated with both gene activities and CpG densities. Most importantly, we constructed the regulatory networks of stage-specific expressed genes and stage-specific activated genes with three omics datasets in bovine early embryos and revealed conserved and distinctive transcriptional regulatory networks between in vivo- and in vitro-derived embryos. This comprehensive analysis revealed critical features of the chromatin landscape and epigenetic reprogramming during bovine early embryo development.

Lauren Gatenby

Papers

The landscape of accessible chromatin in bovine oocytes and early embryos.

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.

The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes.

2 The landscape of accessible chromatin in bovine oocytes and early embryos