Analysis of transcriptional control elements in the 5'-upstream region of ovine interferon-tau gene using feeder-independent caprine trophoblast cell line, HTS-1.

ABSTRACT

Interferon-tau (IFNtau) is a protein secreted from the embryonic trophectoderm of ruminant ungulates during peri-implantation period. This protein acts on the uterine endometrium, which indirectly maintains corpus luteum function, and is therefore considered essential for the process of maternal recognition of pregnancy. Transcriptional regulation of IFNtau genes had been examined using human choriocarcinoma cell lines, JEG-3 or JAR, however, molecular mechanisms by which cell and term specific IFNtau expression are regulated have not been elucidated. Recently, a feeder cell free-trophoblast cell line derived from Shiba-goat placenta, termed HTS-1, was established. In the present investigation, the 5'-upstream region of ovine IFNtau (oIFNtau) gene was analysed using this cell line, which would provide a more suitable system for studies of the ovine trophoblast specific gene than human choriocarcinoma cells. Variously modified 5'-upstream sequences of the oIFNtau gene fused to a luciferase reporter gene were transiently transfected into HTS-1 cells, and human JEG-3 cells were used as a control. These results and co-transfection with expression vectors revealed that Ets-2 binding site in the promoter region was important in HTS-1, whereas AP-1 that binds to the enhancer region was a major activator in JEG-3. By electrophoretic mobility shift assay, a nuclear protein from HTS-1 cells was confirmed to bind specifically to the Ets-2 site of oIFNtau promoter region. Differences in amounts of AP-1 and Ets-2 protein were demonstrated in nuclear extracts from HTS-1, JEG-3 and ovine conceptuses. Substantial differences on oIFNtau gene transcriptions found between caprine HTS-1 and human JEG-3 cells suggest that this cell line could be valuable in the elucidation of a molecular mechanism(s) by which oIFNtau gene expression is regulated in a cell specific manner.