scholarly journals Genome-Wide Mapping of Cytosine Methylation Revealed Dynamic DNA Methylation Patterns Associated with Sporophyte Development of Saccharina japonica

2021 ◽  
Vol 22 (18) ◽  
pp. 9877
Author(s):  
Xiaoqi Yang ◽  
Xiuliang Wang ◽  
Jianting Yao ◽  
Delin Duan
Keyword(s):  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Cytosine Methylation ◽  
Saccharina Japonica ◽  
Transcript Abundance ◽  
Methylation Pattern ◽  
Brown Macroalgae ◽  
Global Methylation ◽  
Genome Wide ◽  
Sporophyte Development

Cytosine methylation plays vital roles in regulating gene expression and plant development. However, the function of DNA methylation in the development of macroalgae remains unclear. Through the genome-wide bisulfite sequencing of cytosine methylation in holdfast, stipe and blade, we obtained the complete 5-mC methylation landscape of Saccharina japonica sporophyte. Our results revealed that the total DNA methylation level of sporophyte was less than 0.9%, and the content of CHH contexts was dominant. Moreover, the distribution of CHH methylation within the genes exhibited exon-enriched characteristics. Profiling of DNA methylation in three parts revealed the diverse methylation pattern of sporophyte development. These pivotal DMRs were involved in cell motility, cell cycle and cell wall/membrane biogenesis. In comparison with stipe and blade, hypermethylation of mannuronate C5-epimerase in holdfast decreased the transcript abundance, which affected the synthesis of alginate, the key component of cell walls. Additionally, 5-mC modification participated in the regulation of blade and holdfast development by the glutamate content respectively via glutamine synthetase and amidophosphoribosyl transferase, which may act as the epigenetic regulation signal. Overall, our study revealed the global methylation characteristics of the well-defined holdfast, stipe and blade, and provided evidence for epigenetic regulation of sporophyte development in brown macroalgae.

scholarly journals Altered DNA methylation pattern reveals epigenetic regulation of Hox genes in thoracic aortic dissection and serves as a biomarker in disease diagnosis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Peiru Liu ◽  
Jing Zhang ◽  
Duo Du ◽  
Dandan Zhang ◽  
Zelin Jin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Aortic Dissection ◽  
Epigenetic Regulation ◽  
Heart Development ◽  
Type A ◽  
Methylation Pattern ◽  
Healthy Controls ◽  
Global Methylation ◽  
Thoracic Aortic Dissection

Abstract Background Thoracic aortic dissection (TAD) is a severe disease with limited understandings in its pathogenesis. Altered DNA methylation has been revealed to be involved in many diseases etiology. Few studies have examined the role of DNA methylation in the development of TAD. This study explored alterations of the DNA methylation landscape in TAD and examined the potential role of cell-free DNA (cfDNA) methylation as a biomarker in TAD diagnosis. Results Ascending aortic tissues from TAD patients (Stanford type A; n = 6) and healthy controls (n = 6) were first examined via whole-genome bisulfite sequencing (WGBS). While no obvious global methylation shift was observed, numerous differentially methylated regions (DMRs) were identified, with associated genes enriched in the areas of vasculature and heart development. We further confirmed the methylation and expression changes in homeobox (Hox) clusters with 10 independent samples using bisulfite pyrosequencing and quantitative real-time PCR (qPCR). Among these, HOXA5, HOXB6 and HOXC6 were significantly down-regulated in TAD samples relative to controls. To evaluate cfDNA methylation pattern as a biomarker in TAD diagnosis, cfDNA from TAD patients (Stanford type A; n = 7) and healthy controls (n = 4) were examined by WGBS. A prediction model was built using DMRs identified previously from aortic tissues on methylation data from cfDNA. Both high sensitivity (86%) and specificity (75%) were achieved in patient classification (AUC = 0.96). Conclusions These findings showed an altered epigenetic regulation in TAD patients. This altered epigenetic regulation and subsequent altered expression of genes associated with vasculature and heart development, such as Hox family genes, may contribute to the loss of aortic integrity and TAD pathogenesis. Additionally, the cfDNA methylation in TAD was highly disease specific, which can be used as a non-invasive biomarker for disease prediction.

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

2007 ◽  
Vol 30 (4) ◽  
pp. 90
Author(s):  
Kirsten Niles ◽  
Sophie La Salle ◽  
Christopher Oakes ◽  
Jacquetta Trasler
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Chromosome 9 ◽  
Specific Gene ◽  
Global Methylation ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

scholarly journals Methylome and transcriptome analyses of soybean response to bean pyralid larvae

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wei-Ying Zeng ◽  
Yu-Rong Tan ◽  
Sheng-Feng Long ◽  
Zu-Dong Sun ◽  
Zhen-Guang Lai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Protein Biosynthesis ◽  
Cell Structure ◽  
Methylation Profile ◽  
Primary Metabolism ◽  
Global Methylation ◽  
Genome Wide ◽  
Differentially Methylated Genes ◽  
Dna Methylation Profile ◽  
Soybean Resistance

Abstract Background Bean pyralid is one of the major leaf-feeding insects that affect soybean crops. DNA methylation can control the networks of gene expressions, and it plays an important role in responses to biotic stress. However, at present the genome-wide DNA methylation profile of the soybean resistance to bean pyralid has not been reported so far. Results Using whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq), we analyzed the highly resistant material (Gantai-2-2, HRK) and highly susceptible material (Wan82–178, HSK), under bean pyralid larvae feeding 0 h and 48 h, to clarify the molecular mechanism of the soybean resistance and explore its insect-resistant genes. We identified 2194, 6872, 39,704 and 40,018 differentially methylated regions (DMRs), as well as 497, 1594, 9596 and 9554 differentially methylated genes (DMGs) in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48 comparisons, respectively. Through the analysis of global methylation and transcription, 265 differentially expressed genes (DEGs) were negatively correlated with DMGs, there were 34, 49, 141 and 116 negatively correlated genes in the HRK0/HRK48, HSK0/HSK48, HSK0/HRK0 and HSK48/HRK48, respectively. The MapMan cluster analysis showed that 114 negatively correlated genes were clustered in 24 pathways, such as protein biosynthesis and modification; primary metabolism; secondary metabolism; cell cycle, cell structure and component; RNA biosynthesis and processing, and so on. Moreover, CRK40; CRK62; STK; MAPK9; L-type lectin-domain containing receptor kinase VIII.2; CesA; CSI1; fimbrin-1; KIN-14B; KIN-14 N; KIN-4A; cytochrome P450 81E8; BEE1; ERF; bHLH25; bHLH79; GATA26, were likely regulatory genes involved in the soybean responses to bean pyralid larvae. Finally, 5 DMRs were further validated that the genome-wide DNA data were reliable through PS-PCR and 5 DEGs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. The results showed an excellent agreement with deep sequencing. Conclusions Genome-wide DNA methylation profile of soybean response to bean pyralid was obtained for the first time. Several specific DMGs which participated in protein kinase, cell and organelle, flavonoid biosynthesis and transcription factor were further identified to be likely associated with soybean response to bean pyralid. Our data will provide better understanding of DNA methylation alteration and their potential role in soybean insect resistance.

scholarly journals DNA hypermethylation associated with upregulated gene expression in prostate cancer demonstrates the diversity of epigenetic regulation

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Ieva Rauluseviciute ◽  
Finn Drabløs ◽  
Morten Beck Rye
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Normal Tissue ◽  
Expression Profiles ◽  
Expression Data ◽  
Dna Hypermethylation ◽  
Genome Wide ◽  
A Genome

Abstract Background Prostate cancer (PCa) has the highest incidence rates of cancers in men in western countries. Unlike several other types of cancer, PCa has few genetic drivers, which has led researchers to look for additional epigenetic and transcriptomic contributors to PCa development and progression. Especially datasets on DNA methylation, the most commonly studied epigenetic marker, have recently been measured and analysed in several PCa patient cohorts. DNA methylation is most commonly associated with downregulation of gene expression. However, positive associations of DNA methylation to gene expression have also been reported, suggesting a more diverse mechanism of epigenetic regulation. Such additional complexity could have important implications for understanding prostate cancer development but has not been studied at a genome-wide scale. Results In this study, we have compared three sets of genome-wide single-site DNA methylation data from 870 PCa and normal tissue samples with multi-cohort gene expression data from 1117 samples, including 532 samples where DNA methylation and gene expression have been measured on the exact same samples. Genes were classified according to their corresponding methylation and expression profiles. A large group of hypermethylated genes was robustly associated with increased gene expression (UPUP group) in all three methylation datasets. These genes demonstrated distinct patterns of correlation between DNA methylation and gene expression compared to the genes showing the canonical negative association between methylation and expression (UPDOWN group). This indicates a more diversified role of DNA methylation in regulating gene expression than previously appreciated. Moreover, UPUP and UPDOWN genes were associated with different compartments — UPUP genes were related to the structures in nucleus, while UPDOWN genes were linked to extracellular features. Conclusion We identified a robust association between hypermethylation and upregulation of gene expression when comparing samples from prostate cancer and normal tissue. These results challenge the classical view where DNA methylation is always associated with suppression of gene expression, which underlines the importance of considering corresponding expression data when assessing the downstream regulatory effect of DNA methylation.

scholarly journals Dynamics of the Methylome and Transcriptome during the Regeneration of Rice

Epigenomes ◽  
2018 ◽  
Vol 2 (3) ◽  
pp. 14 ◽  
Author(s):  
Fei-Man Hsu ◽  
Moloya Gohain ◽  
Archana Allishe ◽  
Yan-Jiun Huang ◽  
Jo-Ling Liao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Oryza Sativa ◽  
Somaclonal Variation ◽  
Cytosine Methylation ◽  
Callus Formation ◽  
Expression Profiles ◽  
Close Association ◽  
Global Methylation ◽  
Regeneration Efficiency ◽  
The Impact

Oryza sativa indica (cv. IR64) and Oryza sativa japonica (cv. TNG67) vary in their regeneration efficiency. Such variation may occur in response to cultural environments that induce somaclonal variation. Somaclonal variations may arise from epigenetic factors, such as DNA methylation. We hypothesized that somaclonal variation may be associated with the differential regeneration efficiency between IR64 and TNG67 through changes in DNA methylation. We generated the stage-associated methylome and transcriptome profiles of the embryo, induced calli, sub-cultured calli, and regenerated calli (including both successful and failed regeneration) of IR64 and TNG67. We found that stage-associated changes are evident by the increase in the cytosine methylation of all contexts upon induction and decline upon regeneration. These changes in the methylome are largely random, but a few regions are consistently targeted at the later stages of culture. The expression profiles showed a dominant tissue-specific difference between the embryo and the calli. A prominent cultivar-associated divide in the global methylation pattern was observed, and a subset of cultivar-associated differentially methylated regions also showed stage-associated changes, implying a close association between differential methylation and the regeneration programs of these two rice cultivars. Based on these findings, we speculate that the differential epigenetic regulation of stress response and developmental pathways may be coupled with genetic differences, ultimately leading to differential regeneration efficiency. The present study elucidates the impact of tissue culture on callus formation and delineates the impact of stage and cultivar to determine the dynamics of the methylome and transcriptome in culture.

scholarly journals The DNA methylation level against the background of the genome size and t-heterochromatin content in some species of the genusSecale L

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2889 ◽  
Author(s):  
Anna Kalinka ◽  
Magdalena Achrem ◽  
Paulina Poter
Keyword(s):  
Dna Methylation ◽  
Genome Size ◽  
Methylation Level ◽  
Cytosine Methylation ◽  
Interspecific Variation ◽  
Distinct Species ◽  
Methylation Pattern ◽  
Biotic And Abiotic Stress ◽  
Complex Issue ◽  
Genomic Scale

Methylation of cytosine in DNA is one of the most important epigenetic modifications in eukaryotes and plays a crucial role in the regulation of gene activity and the maintenance of genomic integrity. DNA methylation and other epigenetic mechanisms affect the development, differentiation or the response of plants to biotic and abiotic stress. This study compared the level of methylation of cytosines on a global (ELISA) and genomic scale (MSAP) between the species of the genusSecale. We analyzed whether the interspecific variation of cytosine methylation was associated with the size of the genome (C-value) and the content of telomeric heterochromatin. MSAP analysis showed thatS. sylvestrewas the most distinct species among the studied rye taxa; however, the results clearly indicated that these differences were not statistically significant. The total methylation level of the studied loci was very similar in all taxa and ranged from 60% inS. strictumssp.africanumto 66% inS. cerealessp.segetale, which confirmed the lack of significant differences in the sequence methylation pattern between the pairs of rye taxa. The level of global cytosine methylation in the DNA was not significantly associated with the content of t-heterochromatin and did not overlap with the existing taxonomic rye relationships. The highest content of 5-methylcytosine was found inS. cerealessp.segetale(83%), while very low inS. strictumssp.strictum(53%), which was significantly different from the methylation state of all taxa, except forS. sylvestre. The other studied taxa of rye had a similar level of methylated cytosine ranging from 66.42% (S. vavilovii) to 74.41% in (S. cerealessp.afghanicum). The results obtained in this study are evidence that the percentage of methylated cytosine cannot be inferred solely based on the genome size or t-heterochromatin. This is a significantly more complex issue.

scholarly journals Genome-Wide DNA Methylation Variations Between Grass Carp with Different Ages

2020 ◽  
Author(s):  
Libo He ◽  
Denghui Zhu ◽  
Pengfei Chu ◽  
Yongming Li ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Immune Response ◽  
Grass Carp ◽  
Energy Production ◽  
Gene Body ◽  
Global Methylation ◽  
Genome Wide ◽  
Age Dependent ◽  
Different Ages

Abstract Background: Grass carp is an important farmed fish in China that infected by many pathogens, especially grass carp reovirus (GCRV). Notably, grass carp showed age-dependent susceptibility to GCRV, while the mechanism remains unclear. Herein, we performed a genome-wide survey of differences in DNA methylation and gene expression between five months old grass carp (FMO, sensitive to GCRV) and three years old grass carp (TYO, resistant to GCRV) aim to uncover the mechanism.Results: Colorimetric quantification revealed global methylation level of TYO fish was higher than that of FMO fish. Whole-genome bisulfite sequencing (WGBS) of two groups revealed 6,214 differentially methylated regions (DMRs) and 4,052 differentially methylated genes (DMGs), with most of DMRs and DMGs showed hypermethylation patterns in TYO fish. Correlation analysis indicated that DNA hypomethylation in promoter negative correlated with gene expression, whereas positive correlation was found between gene-body DNA hypermethylation and gene expression. Enrichment analysis revealed that promoter hypo-DMGs in TYO fish were significant enriched in pathways involved in immune response while gene-body hyper-DMGs in TYO fish were significant enriched in terms related to RNA transcription, biosynthetic, and energy production. RNA-seq indicated these terms or pathways involved in immune response, biosynthetic, and energy production also significant enriched for the up-regulated genes in TYO fish. Conclusions: Collectively, these results revealed the genome-wide DNA methylation variations between grass carp with different ages. DNA methylation and gene expression variations in genes involved in immune response, biosynthetic, and energy production may contributed to the age-dependent susceptibility to GCRV in grass carp. Our results will provide important information for the disease-resistant breeding programs of grass carp and may also benefit to the research of age-dependent diseases in human.

scholarly journals High Resolution Genome Wide DNA Methylation Analysis in a Large Trial Group Reveals a Novel Epigenetically Defined Subgroup of Myeloma Patients Characterized By Developmental Gene Hypermethylation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2189-2189
Author(s):  
Martin F Kaiser ◽  
Alexander Murison ◽  
Charlotte Pawlyn ◽  
Eileen M Boyle ◽  
David C Johnson ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
Methylation Pattern ◽  
Gene Set Enrichment Analysis ◽  
Epigenetic Changes ◽  
Dna Hypomethylation ◽  
Dna Hypermethylation ◽  
Epigenetic Programming ◽  
Genome Wide ◽  
Dna Methylation Pattern

Abstract Introduction Multiple myeloma is a clinically highly heterogeneous disease, which is reflected by both a complex genome and epigenome. Dynamic epigenetic changes are involved at several stages of myeloma biology, such as transformation and disease progression. Our previous genome wide epigenetic analyses identified prognostically relevant DNA hypermethylation at specific tumor suppressor genes (Kaiser MF et al., Blood 2013), indicating that specific epigenetic programming influences clinical behavior. This clinically relevant finding prompted further investigation of the epigenomic structure of myeloma and its interaction with genetic aberrations. Material and Methods Genome wide DNA methylation of CD138-purified myeloma cells from 464 patients enrolled in the NCRI Myeloma XI trial at presentation were analyzed using the high resolution 450k DNA methylation array platform (Illumina). In addition, 4 plasma cell leukemia (PCL) cases (two t(11;14) and two (4;14)) and 7 myeloma cell lines (HMCL) carrying different translocations were analysed. Analyses were performed in R Bioconductor packages after filtering and removal of low quality and non-uniquely mapping probes. Results Variation in genome wide DNA methylation was analyzed using unsupervised hierarchical clustering of the 10,000 most variable probes, which revealed epigenetically defined subgroups of disease. Presence of recurrent IGH translocations was strongly associated with specific epigenetic profiles. All 60 cases with t(4;14) clustered into two highly similar sub-clusters, confirming that overexpression of the H3K36 methyltransferase MMSET in t(4;14) has a defined and specific effect on the myeloma epigenome. Interestingly, HMCLs KMS-11 and LP-1, which carry t(4;14), MM1.S, a t(14;16) cell line with an E1099K MMSET activating mutation as well as two PCLs with t(4;14) all clustered in one sub-clade. The majority (59/85) of t(11;14) cases showed global DNA hypomethylation compared to t(4;14) cases and clustered in one subclade, indicating a epigenetic programming effect associated with CCND1, with a subgroup of t(11;14) cases showing a variable DNA methylation pattern. In addition to translocation-defined subgroups, a small cluster of samples with a distinct epigenetic profile was identified. In total 7 cases with a shared specific DNA methylation pattern (median inter-sample correlation 0.4) were identified. The group was characterized by DNA hypermethylation (4,341 hypermethylated regions vs. 750 hypomethylated regions) in comparison to all other cases. Intersection of regions hypermethylated in this subgroups with ENCODE datasets revealed mapping to poised enhancers and promoters in H1-hESC, indicating functionally relevant epigenetic changes. Gene set enrichment analysis (KEGG) demonstrated enrichment of developmental pathway genes, e.g. Hedgehog signaling (adj p=5x10exp-13), amongst others and all four HOX clusters were differentially methylated in this group. Of note, three of seven cases in this subgroup carried a t(11;14) and all t(11;14) or t(11;14)-like HMCLs clustered closely together with these patient cases, but not with the cluster carrying the majority of t(11;14) myeloma or t(11;14) PCLs. This potentially indicates that t(11;14) HMCL could be derived from a subgroup of patients with specific epigenetic characteristics. Conclusion Our results indicate that the recurrent IGH translocations are fundamentally involved in shaping the myeloma epigenome through either direct upregulation of epigenetic modifiers (e.g. MMSET) or through insufficiently understood mechanisms. However, developmental epigenetic processes seem to independently contribute to the complexity of the epigenome in some cases. This work provides important insights into the spectrum of epigenetic subgroups of myeloma and helps identify subgroups of disease that may benefit from specific epigenetic therapies currently being developed. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.

scholarly journals Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members

2020 ◽  
Vol 48 (8) ◽  
pp. 4081-4099 ◽  
Author(s):  
Alex I Finnegan ◽  
Somang Kim ◽  
Hu Jin ◽  
Michael Gapinske ◽  
Wendy S Woods ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Time Course ◽  
Cytosine Methylation ◽  
Family Members ◽  
Dynamic Network ◽  
Dna Methyltransferases ◽  
Genome Wide ◽  
Distinct Sequence ◽  
Adenosyl Methionine ◽  
Methylation Patterns

Abstract Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 126
Author(s):  
J. E. Duan ◽  
Z. Jiang ◽  
F. Alqahtani ◽  
I. Mandoiu ◽  
H. Dong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Epigenetic Regulation ◽  
Bisulfite Sequencing ◽  
Whole Genome ◽  
Differentially Methylated Regions ◽  
Whole Genome Bisulfite Sequencing ◽  
Cpg Sites ◽  
Genome Wide ◽  
Genome Bisulfite Sequencing

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

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