Expression of ovine interferon-tau (oIFNτ) genes, essential for the maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast and is not detected in any other cell types or tissues. Substantial secretion of oIFNτ starts on day 12-13 of pregnancy (day 0 = day of estrus), reaches the highest on day 16-17, and then declines rapidly. Ovine IFNτ mRNA, on the other hand, reaches the highest level on day 14 of pregnancy, 2-3 days before peak production of the protein. In this study, day 14 and 17 conceptuses treated with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation, were cultured in vitro and only day 17, not day 14, conceptuses resulted in upregulation of oIFNτ gene expression. To gain insight into the molecular mechanism of oIFNτ gene downregulation, the methylation status within 1 kb of the 5′-flanking region of oIFNτ-o10 gene was investigated: CpG dinucleotides of this gene in day 14 ovine conceptuses were hypomethylated compared to day 20 conceptuses or other tissues. In vitro methylation of oIFNτ-o10-reporter constructs caused suppression of reporter activity in transient transfections. Cotransfection of methyl-CpG-binding protein (MeCP2) with the reporter construct elicited further suppression of the reporter activity. In electrophoretic mobility shift assay (EMSA), patterns of shifted bands did not show much difference between methylated and unmethylated probes in distal regions, but exhibited differences in the proximal region of upstream sequences of the OIFNτ gene. These results provide evidence that changes in the degree of DNA methylation could be one of the major mechanisms leading to downregulation of the oIFNτ-o10 gene during early gestation, and possibly its silencing in nonconceptus tissues.
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