Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/1999
Publication Date: N/A
Citation: Interpretive Summary: Reproductive loss during early pregnancy is one of the most costly factors influencing the livestock industry. A large majority of reproductive failure in sheep occurs as a result of an impaired signal from the developing conceptus to the maternal system during implantation. In sheep, the gene controlling the conceptus signaling product (ovine interferon-tau; ;oIFNt) is known. The objective of this study was to determine specific regions within the gene that control expression of the signaling product in an uterine cell culture system. Results indicate that upstream regions from -654 to -607 bases and from -606 to -555 bases were essential for oIFNt gene expression. Enhancer and silencer regions of the gene were determined. Conceptus gene expression controls maternal recognition of pregnancy and implantation and can increase embryo survival and improve lambing rates for the U.S. sheep industry.
Technical Abstract: To examine regulatory mechanisms of ovine interferon-tau (oIFNt) gene expression, potential enhancer/silencer elements of the oIFNt gene were examined using a transient transfection system with oIFNt gene- chloramphenicol acetyltransferase reporter (oIFNt-CAT) constructs in human choriocarcinoma cells, JEG3. First, the experiments with 5'-deletion constructs revealed that the upstream regions from -654 to -607 bases and from -606 to -555 bases were essential for oIFNt gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNt were inserted in front of SV40 promoter, the regions between -654 and -555 bases were determined to be the enhancer region required for oIFNt-SV40-CAT transactivation. A subsequent study with the oIFNt-CAT constructs lacking the upstream region between -542 and -124 bases revealed that, in addition to the further upstream region between -1000 and -654 bases, the sequences from -543 to -452 bases seemed to act as silencer regions. The oIFNt-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between -654 and -555 bases of the oIFNt gene. Based on nucleotide sequence analysis, there are numerous sites between -654 and -555 bases to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, c-jun plus c-fos expression enhanced oIFNt-CAT transactivation, however, GATA-1, -2, or -3 expression did not. Taken together, these results suggest that the upstream region between -654 and -555 bases could be considered as the enhancer region of oIFNt gene transactivation.