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Increase in DNA methylation downregulates conceptus interferon-tau gene expression
Hisashi Nojima1, Kentaro Nagaoka, Ronald K Christenson
1Laboratory of Animal Breeding, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan.
Molecular Reproduction and Development
|March 3, 2004
Summary
DNA methylation regulates ovine interferon-tau (oIFNtau) gene expression during early pregnancy. Hypomethylation in early conceptuses contrasts with later downregulation, suggesting methylation
Area of Science:
- Reproductive Biology
- Epigenetics
- Molecular Biology
Background:
- Ovine interferon-tau (oIFNtau) is crucial for pregnancy recognition in ruminants.
- oIFNtau expression is trophoblast-specific and follows a distinct temporal pattern during early gestation.
- Understanding oIFNtau gene regulation is key to reproductive success in sheep.
Purpose of the Study:
- To investigate the role of DNA methylation in the downregulation of ovine interferon-tau (oIFNtau) gene expression.
- To elucidate the molecular mechanisms underlying oIFNtau gene silencing during early gestation.
- To examine the methylation status of the oIFNtau gene in different gestational stages and tissues.
Main Methods:
- In vitro culture of ovine conceptuses treated with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor.
- Analysis of the methylation status of the 5'-flanking region of the oIFNtau-o10 gene using bisulfite sequencing.
- Reporter gene assays to assess the impact of DNA methylation on oIFNtau promoter activity.
- Electrophoretic mobility shift assays (EMSA) to study protein binding to methylated and unmethylated DNA probes.
Main Results:
- Treatment with a DNA methylation inhibitor upregulated oIFNtau expression in day 17, but not day 14, conceptuses.
- CpG dinucleotides in the 5'-flanking region of the oIFNtau-o10 gene were hypomethylated in day 14 conceptuses compared to day 20 conceptuses and other tissues.
- In vitro methylation suppressed reporter gene activity, and MeCP2 cotransfection further reduced it.
- EMSA revealed differences in protein binding to methylated vs. unmethylated probes in the proximal upstream region of the oIFNtau gene.
Conclusions:
- Changes in DNA methylation are a significant mechanism for downregulating oIFNtau gene expression during early gestation.
- DNA methylation may contribute to the silencing of the oIFNtau gene in non-conceptus tissues.
- These findings provide insights into the epigenetic regulation of pregnancy recognition in ruminants.