To examine regulatory mechanisms of sheep interferon τ (oIFNτ) gene expression, potential enhancer/silencer elements of the oIFNτ gene were examined using a transient transfection system with oIFNτ gene-chloramphenicol acetyltransferase (oIFNτ-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5ʹ-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNτ gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNτ were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNτ-SV40-CAT transactivation. A subsequent study with the oIFNτ-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNτ-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNτ gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 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, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNτ-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNτ gene transactivation.
- CAT reporter gene
- enhancer elements
- interferon gene expression
- The Biochemical Society, London © 1999