STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes.

The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter.     

The promoter of the human gene for insulin-like growth factor binding protein-1. Basal promoter activity in HEP G2 cells depends upon liver factor B1.

Characterization of the human insulin-like growth factor binding protein-1 (IGFBP-1) promoter was initiated to facilitate study of developmental and hormonal factors regulating IGFBP-1 production. The region immediately 5' to the IGFBP-1 mRNA capsite is typical of a eukaryotic promoter, with a TATA sequence beginning 28 base pairs (bp) and a CCAAT promoter element beginning 72 bp upstream from this capsite. A 1.3-kilobase insert containing the IGFBP-1 capsite and 1205 bp of this putative IGFBP-1 promoter region directs expression of the reporter gene chloramphenicol acetyltransferase (CAT) in an orientation-specific manner in transfected HEP G2 cells, and the capsite identified for the CAT mRNA is identical to that identified for native IGFBP-1 mRNA. These observations suggest that the 1.3-kilobase insert contains the IGFBP-1 promoter. This promoter was further characterized by deletion analysis, site-directed mutagenesis, gel mobility shift assays, and DNaseI protection assays. These studies identify the CCAAT box region as the major cis element involved in basal IGFBP-1 promoter activity in HEP G2 cells, demonstrate that increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region, and indicate that the DNA binding factor(s) responsible for increased basal promoter activity is related to liver factor B1. These observations suggest that liver B1 is the major trans-acting factor stimulating basal IGFBP-1 promoter activity in HEP G2 cells.     

The CCAAT-binding factor CBF/NF-Y regulates the human acetylcholinesterase promoter activity during calcium ionophore A23187-induced cell apoptosis

We previously reported that the expression of acetylcholinesterase during A23187-induced apoptosis of HeLa cells is regulated by Ca(2+) mobilization through the modulation of mRNA stability and acetylcholinesterase promoter activity. Transactivation of the human acetylcholinesterase promoter by A23187 was partially mediated by the distal CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter, which was bound by the CCAAT binding factor (CBF/NF-Y). In the present study, we investigated the molecular mechanisms by which CBF/NF-Y regulates A23187-induced activation of the human acetylcholinesterase promoter. The results indicate that CBF/NF-Y binding to the distal CCAAT motif suppresses the promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that binding of CBF/NF-Y to the distal CCAAT motif decreased after A23187 treatment. Our results suggest that acetylcholinesterase promoter activation during A23187-induced HeLa cell apoptosis may result partly from the dissociation of CBF/NF-Y from the distal CCAAT motif in the acetylcholinesterase promoter, reversing this suppression.