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DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

O'Doherty AM, Magee DA, O'Shea LC, Forde N, Beltman ME, Mamo S, Fair T - BMC Dev. Biol. (2015)

Bottom Line: For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05).Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland. alan.odoherty@ucd.ie.

ABSTRACT

Background: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.

Results: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.

Conclusion: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

No MeSH data available.


Related in: MedlinePlus

Overview of DMR methylation at imprinted genes and expression of associated transcripts in pre-implantation bovine embryos. Schematic representation of DNA methylation imprint dynamics following the blastocyst stage of bovine embryogenesis. Shaded grey area denotes the range of observed methylation values at imprinted loci during pre-implantation embryo development. Solid and dashed lines represents expression profiles of key genes associated with imprinted establishment and maintenance; blue dashed line DNMT3A and DNMT3B, solid orange line DNMT1 and TRIM28/KAP1 and solid orange-dashed orange line ZFP57, UHRF1 family and TET family transcripts. Question marks to the left of the vertical dashed red line denote that technical variation cannot be completely excluded at this stage.
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Fig5: Overview of DMR methylation at imprinted genes and expression of associated transcripts in pre-implantation bovine embryos. Schematic representation of DNA methylation imprint dynamics following the blastocyst stage of bovine embryogenesis. Shaded grey area denotes the range of observed methylation values at imprinted loci during pre-implantation embryo development. Solid and dashed lines represents expression profiles of key genes associated with imprinted establishment and maintenance; blue dashed line DNMT3A and DNMT3B, solid orange line DNMT1 and TRIM28/KAP1 and solid orange-dashed orange line ZFP57, UHRF1 family and TET family transcripts. Question marks to the left of the vertical dashed red line denote that technical variation cannot be completely excluded at this stage.

Mentions: Targeted mining of bovine embryo global transcriptomic data [54] revealed a distinctive temporal RNA transcript profile for several key genes associated with establishing and maintaining imprints (DNMT3A, DNMT3B, DNMT1, TRIM28/KAP1 and ZFP57). High levels of mRNA expression were observed during the period of greatest imprint instability, followed by a significant decrease in transcript abundance at later stages of development, in tandem with DNA methylation imprint stabilization. This pattern of expression was also observed for DNMT3A, DNMT3A2 and DNMT3B, complementing the gene expression profiles. In mice, trim28/kap1 maternal knockouts present with severe phenotypic and epigenetic variability resulting in embryonic lethality [35], it has been proposed that an epigenetic complex formed by TRIM28/KAP1 and ZFP57 facilitate imprint maintenance/protection during the period of pre-implantation reprogramming [68-70]. In addition, the de novo and maintenance DNA methyltransferases, DNMT3A/B and DNMT1, were shown to interact with ZFP57 through its co-factor TRIM28/KAP1 to maintain methylation imprints in embryonic stem cells [36,69]. Taking all things in to consideration, it is likely that TRIM28/KAP1 and ZFP57, along with the DNA methyltransferases DNMT3A, DNMT3B and DNMT1 actively facilitate a window of DNA methylation reprogramming in bovine embryos post blastocyst development (Figure 5).Figure 5


DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

O'Doherty AM, Magee DA, O'Shea LC, Forde N, Beltman ME, Mamo S, Fair T - BMC Dev. Biol. (2015)

Overview of DMR methylation at imprinted genes and expression of associated transcripts in pre-implantation bovine embryos. Schematic representation of DNA methylation imprint dynamics following the blastocyst stage of bovine embryogenesis. Shaded grey area denotes the range of observed methylation values at imprinted loci during pre-implantation embryo development. Solid and dashed lines represents expression profiles of key genes associated with imprinted establishment and maintenance; blue dashed line DNMT3A and DNMT3B, solid orange line DNMT1 and TRIM28/KAP1 and solid orange-dashed orange line ZFP57, UHRF1 family and TET family transcripts. Question marks to the left of the vertical dashed red line denote that technical variation cannot be completely excluded at this stage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363183&req=5

Fig5: Overview of DMR methylation at imprinted genes and expression of associated transcripts in pre-implantation bovine embryos. Schematic representation of DNA methylation imprint dynamics following the blastocyst stage of bovine embryogenesis. Shaded grey area denotes the range of observed methylation values at imprinted loci during pre-implantation embryo development. Solid and dashed lines represents expression profiles of key genes associated with imprinted establishment and maintenance; blue dashed line DNMT3A and DNMT3B, solid orange line DNMT1 and TRIM28/KAP1 and solid orange-dashed orange line ZFP57, UHRF1 family and TET family transcripts. Question marks to the left of the vertical dashed red line denote that technical variation cannot be completely excluded at this stage.
Mentions: Targeted mining of bovine embryo global transcriptomic data [54] revealed a distinctive temporal RNA transcript profile for several key genes associated with establishing and maintaining imprints (DNMT3A, DNMT3B, DNMT1, TRIM28/KAP1 and ZFP57). High levels of mRNA expression were observed during the period of greatest imprint instability, followed by a significant decrease in transcript abundance at later stages of development, in tandem with DNA methylation imprint stabilization. This pattern of expression was also observed for DNMT3A, DNMT3A2 and DNMT3B, complementing the gene expression profiles. In mice, trim28/kap1 maternal knockouts present with severe phenotypic and epigenetic variability resulting in embryonic lethality [35], it has been proposed that an epigenetic complex formed by TRIM28/KAP1 and ZFP57 facilitate imprint maintenance/protection during the period of pre-implantation reprogramming [68-70]. In addition, the de novo and maintenance DNA methyltransferases, DNMT3A/B and DNMT1, were shown to interact with ZFP57 through its co-factor TRIM28/KAP1 to maintain methylation imprints in embryonic stem cells [36,69]. Taking all things in to consideration, it is likely that TRIM28/KAP1 and ZFP57, along with the DNA methyltransferases DNMT3A, DNMT3B and DNMT1 actively facilitate a window of DNA methylation reprogramming in bovine embryos post blastocyst development (Figure 5).Figure 5

Bottom Line: For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05).Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland. alan.odoherty@ucd.ie.

ABSTRACT

Background: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.

Results: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.

Conclusion: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

No MeSH data available.


Related in: MedlinePlus