Different levels of ovine interferon-τ gene expressions are regulated through the short promoter region including Ets-2 binding site

Abstract

Regulation of interferon-tau (IFNtau) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNtau (oIFNtau) gene sequences characterized, approximately 75% of oIFNtau transcripts expressed in utero is derived from oIFNtau-o10 gene and amounts of transcripts from other oIFNtau genes such as oIFNtau-o8 or oIFNtau-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNtau-o10 and oIFNtau-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNtau genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNtau genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNtau-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNtau-o10 gene inserted into the oIFNtau-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNtau-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 oIFNtau-o10 gene was required for oIFNtau-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNtau-o10 and oIFNtau-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 oIFNtau gene expressions seen in utero.