Different Levels of Ovine Interferon-t Gene Expressions Are Regulated Through the Short Promoter Region Including Ets-2 Binding Site

Regulation of interferon-t (IFNt) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNt (oIFNt) gene sequences characterized, approximately 75% of oIFNt transcripts expressed in utero is derived from oIFNt-o10 gene and amounts of transcripts from other oIFNt genes such as oIFNt-o8 or oIFNt-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNt-o10 and oIFNt-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNt genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 50-upstream regions of these oIFNt genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNt-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNt-o10 gene inserted into the oIFNt-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNt-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNt-o10 gene was required for oIFNt-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNt-o10 and oIFNt-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNt gene expressions seen in utero