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Characterization of DNA methylation and transcriptomics during early pregnancy in dairy cows

ABSTRACT: DNA methylation is an essential epigenetic mechanism involved in cell differentiation, proliferation, apoptosis, and metabolism. However, the DNA methylation changes of related genes during early pregnancy in dairy cows remain unclear . The purpose of this study was to study DNA methylation-related changes in early pregnancy cows and construct gene pools associated with pregnancy. In this study, we used peripheral blood samples from 15 healthy, productive and similar cows at 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl) and association analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and Quantitative Real-time PCR (RT-qPCR) method are used to verify the Methylation differential genes. Our findings showed that DNA methylation majorly occurred in the cytosine-guanine (CG) range, and the amount of hypomethylation was higher than that of hypermethylation in 3Utrs, introns and promoters. A total of 76530 differentially methylated sites (DMCs) and 16411 differentially methylated regions (DMRs) were identified. RNA-seq identified 8858 differentially expressed genes (DEGs), with 4499 down-DEGs and 4359 up-DEGs. However, there were 44 significant differentially expressed genes, including 25 up-DEGs and 19 down-DEGs. DMC gene ontology (GO) analysis revealed significant enrichment in developmental process, cell differentitaion and nevous system development in biological processes (BP)；Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer overview and cellular and Gastric cancer and Immune disease signaling pathways. DMRs GO analysis highlighted significant enrichment in the developmental process, cell differentiation and nervous system development; KEGG the results showed that significant enrichment in Signaling pathways regulating pluripotency of stem cells, Systemic lupus erythematosus，Transcriptional misregulation in cancer，Spliceosome，Herpes simplex virus 1 infection, Maturity onset diabetes of the young. DEG GO analysis results show that the main enrichment in BP differentiation of bone marrow cells, red blood cell differentiation and erythrocyte steady-state and anion during transportation and transshipment; KEGG analysis mainly concentrated in retinol metabolism, purine metabolism, cAMP signaling pathway and Gap junction pathway. Through a comprehensive analysis of RRBS and transcriptomics, 12 genes, including HBB, HBA 1, GPR 4, CPXM 2, SLC20A2, AFAP 1 and DOK 7, were different both in the methylome and the transcriptome. Early pregnancy changes in DNA methylation were significant and mainly down-regulated. These results can be the epigenetic changes in early pregnancy for cows provide new insights.

ORGANISM(S): Bos taurus

PROVIDER: GSE274885 | GEO | 2025/08/09

REPOSITORIES: GEO

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Project description:DNA methylation is an essential epigenetic mechanism involved in cell differentiation, proliferation, apoptosis, and metabolism. However, the DNA methylation changes of related genes during early pregnancy in dairy cows remain unclear . The purpose of this study was to study DNA methylation-related changes in early pregnancy cows and construct gene pools associated with pregnancy. In this study, we used peripheral blood samples from 15 healthy, productive and similar cows at 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl) and association analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and Quantitative Real-time PCR (RT-qPCR) method are used to verify the Methylation differential genes. Our findings showed that DNA methylation majorly occurred in the cytosine-guanine (CG) range, and the amount of hypomethylation was higher than that of hypermethylation in 3Utrs, introns and promoters. A total of 76530 differentially methylated sites (DMCs) and 16411 differentially methylated regions (DMRs) were identified. RNA-seq identified 8858 differentially expressed genes (DEGs), with 4499 down-DEGs and 4359 up-DEGs. However, there were 44 significant differentially expressed genes, including 25 up-DEGs and 19 down-DEGs. DMC gene ontology (GO) analysis revealed significant enrichment in developmental process, cell differentitaion and nevous system development in biological processes (BP);Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer overview and cellular and Gastric cancer and Immune disease signaling pathways. DMRs GO analysis highlighted significant enrichment in the developmental process, cell differentiation and nervous system development; KEGG the results showed that significant enrichment in Signaling pathways regulating pluripotency of stem cells, Systemic lupus erythematosus，Transcriptional misregulation in cancer，Spliceosome，Herpes simplex virus 1 infection, Maturity onset diabetes of the young. DEG GO analysis results show that the main enrichment in BP differentiation of bone marrow cells, red blood cell differentiation and erythrocyte steady-state and anion during transportation and transshipment; KEGG analysis mainly concentrated in retinol metabolism, purine metabolism, cAMP signaling pathway and Gap junction pathway. Through a comprehensive analysis of RRBS and transcriptomics, 12 genes, including HBB, HBA 1, GPR 4, CPXM 2, SLC20A2, AFAP 1 and DOK 7, were different both in the methylome and the transcriptome. Early pregnancy changes in DNA methylation were significant and mainly down-regulated. These results can be the epigenetic changes in early pregnancy for cows provide new insights.

Project description:[Background] DNA methylation plays a crucial role in regulating cell proliferation, differentiation, gene expression, and embryonic development. However, the comprehensive changes in genome-wide DNA methylation during early pregnancy in sows remain unknown. This study aimed to investigate the DNA methylation profiles and to construct an early gene pool of sows during early gestation.[Results] In this investigation, we utilized blood samples from 20 healthy sows and applied Reduced Representation Bisulfite Sequencing (RBSS) technology to systematically examine alterations in DNA methylation in sow blood during early pregnancy. Screening and validating the Methylation differential genes by using bisulfite sequencing PCR (BSP) and Quantitative Real-time PCR (RT-qPCR). Our findings indicated a significant increase in methylation levels on day 28 of pregnancy (pre28) compared to non-pregnancy (ctrl) across various gene elements, including CGI, promoter, and genebody. A total of 64,908 differentially methylated sites (DMCs) and 12,119 differentially methylated regions (DMRs) were identified, predominantly exhibiting methylation upregulation. Randomly selected five differentially methylated genes—VBP1, UPRT (upregulated methylation), RPL10, RAB33A, and CD93 (downregulated methylation)—were validated. Between the pregnant (pre28) and non-pregnant (ctrl) groups the BSP results showed highly significant methylation differences (P<0.01) in VBP1, UPRT, RPL10, and RAB33A. DMC gene ontology (GO) analysis revealed significant enrichment in nuclear receptor activity, negative regulation of cysteine-type endopeptidase activity during apoptosis, heme binding, and hormonal activity. DMRs GO analysis highlighted significant enrichment in 1-acylglycerol-3-phosphate O-acyltransferase activity, regulation of transforming growth factor β receptor signaling pathway, mitochondrial outer membrane and myotubular cell development, and cellular calcium homeostasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in the MAPK signaling pathway and the PI3K-Akt signaling pathway.[Conclusion] During sow early gestation, DNA methylation is significant changes and is mainly upregulated. The observed upregulation of DNA methylation during early gestation in sows may regulate the MAPK and PI3K-Akt signaling pathways, potentially influencing embryonic development. Methylated differential genes VBP1 and RPL10 can be used as potential markers for early sow pregnancy diagnosis. These findings offer novel insights into the epigenetic changes occurring in sows during early gestation.

Project description:Co-ordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A non-receptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. To gain support for this hypothesis the correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and sub-fertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with transcription profiles which have been previously reported. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. The 1,000 most significant correlations were used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when comparing pregnant and cycling animals and 11% were differentially expressed comparing pregnant fertile and sub-fertile animals. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies. The estrous cycles of 24 lactating dairy cows were synchronized (at 58.8 (SEM 3.77) and 60.2 (SEM 1.51) days post calving in dairy cows of sub-fertile and fertile strains, respectively) and 14 received a single embryo transferred on day 7 of the estrous cycle. Animals were slaughtered at day 17 of the reproductive cycle and endometrial tissues (both caruncular and intercaruncular) were sampled. Selection criteria for the study included strain and calving date, and health postcalving was an exclusion criterion (cows with severe uterine infections or mastitis were excluded before being enrolled in the embryo transfer round). Cows in each strain were matched for calving number and age. A total of 10 cycling and 12 pregnant animals enrolled in the study were utilized, due to the associated costs of slaughtering the cows. These animals represented fertile (six pregnant and five cycling Holstein-Friesian cows with New Zealand ancestry/M-bM-^IM-$30% North American genetics, n=11, NZ) and sub-fertile (six pregnant and five cycling Holstein-Friesian cows with >87% North American ancestry, n=11, NA) phenotypes of Holstein-Friesian dairy cows

Project description:Post-partum uterine inflammation (endometritis) is associated with lower fertility at both the time of infection and after the inflammation has resolved. It was hypothesized that aberrant DNA methylation may be involved in the sub-fertility associated with post-partum uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with sub-clinical endometritis. Endometrial tissues were obtained at 29 days post-partum (n=12) and Agilent two-colour microarrays were used to characterize transcription and DNA methylation profiles. Analyses revealed 1,856 probes to be differentially expressed in animals with subclinical endometritis (SUI, n=6) compared with control cows (NUI, n=6, P<0.05, Storey Multiple testing correction). No significant associations among DNA methylation and gene expression were detected. Further analysis of gene expression data using GeneGo Metacore and Gene Set Enrichment Analysis identified several pathways and processes enriched in the comparison. Several pathways that are involved in the innate immune response were enriched in SUI cows. Consistent with the presence of microorganisms in the uterus, there was enrichment for the Toll-like receptor (TLR) signaling pathway, including increased expression of the transcription factor NFKB1, the pro-inflammatory cytokines IL1A and IL1B, downstream chemokines, cytokines, and acute phase and antimicrobial proteins in the endometrium of SUI cows. Furthermore, the chemokine signaling pathway was enriched in SUI cows, with increased expression of genes that attract cells of the innate immune system. Increased expression of IL-8 and CXCL6, chemotactic factors for recruitment of neutrophils along with the immune cell surface marker PTPRC in SUI cows is consistent with the greater number of polymorphonuclear cells present in the uterus of these cows. Several antimicrobial peptides (LAP, TAP, DEFB1, DEFB10, DEFB103B, DEFB7) and acute phase proteins, including SAA3, LBP, and the complement gene CFB, had greater expression in SUI cows. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local pro-inflammatory environment in the uterus. If this period of inflammation is prolonged, it could result in tissue damage or failure to complete involution of the uterus, which may create a sub-optimal environment for future pregnancy. Agilent two-colour microarrays were used to characterize DNA methylation profiles in cows with subclinical endometritis (SUI, n=6) compared to control cows (NUI, n=6). Endometrial tissues (caruncular, intercaruncular) were obtained at 29 days post-partum.

Project description:To determine the effects of CNN2 knockdown on the molecular phenotype of NSCLC cells, global RNA-Seq-based transcriptomic analysis was performed in parallel with global tandem mass tag multiplexing-based proteomic analysis in A549 cells transduced with shCtrl or shCNN2 . Knockdown of CNN2 resulted in 3,980 DEGs and 539 DEPs in A549 cells (Limma-moderated t-test). Venn analysis of these DEPs and DEGs revealed 222 differentially expressed targets at both mRNA and protein levels. After which, the DEGs and DEPs were mapped based on their log2-fold change values to profile the consistency of up- or downregulation at mRNA and protein levels. This mapping revealed four distinct groups of differentially expressed targets: (i) The ‘same’ group represents differentially expressed targets with the same trend at the mRNA level and protein level; (ii) the ‘opposite’ group represents differentially expressed targets with an opposite trend at the mRNA level and protein level; (iii) the ‘transcript_only’ group represents differentially expressed targets at the mRNA level but not at the protein level; and (iv) the ‘protein_only’ group represents differentially expressed targets at the protein level but not at the mRNA level. To improve understanding of the biological relevance of DEGs and DEPs, functional enrichment analysis was performed on GO and KEGG pathway enrichment analysis. The GO analysis revealed significant enrichment for several GO-BP, including wound healing, DNA replication, neutrophil degranulation and mitotic cytokinesis; several GO-MF, including protein domain-specific binding, cadherin binding and actin binding; and several GO-CC, including focal adhesion, membrane raft and centrosome . Breaking down the GO-BP analysis by group, it was found that the ‘same’ group consistently showed significant enrichment for neutrophil degranulation, wound healing, DNA replication and mitotic cytokinesis . KEGG analysis revealed significant enrichment for several KEGG pathways, including proteoglycans in cancer, actin cytoskeleton regulation and cell cycle. Partitioning the KEGG analysis by group revealed that the ‘same’ group consistently showed significant enrichment for proteoglycans in cancer, actin cytoskeleton regulation and cell cycle . Consistent with the role of CNN2 as a regulatory actin-binding protein, these combined results suggest that CNN2 may play a role in regulating actin cytoskeleton-mediated DNA replication and cell cycle progression in NSCLC cells.

Project description:The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endo- metria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status. mRNA profiles of pregnant vs not pregnant cross-bred beef heifers at days 13 and 16 (n=5 per group)

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Characterization of DNA methylation and transcriptomics during early pregnancy in dairy cows

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