Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.

We have compared the activities of mouse alpha-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). MersI, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single trans-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Localization of DNA protein-binding sites in the proximal and distal promoter regions of the mouse alpha-fetoprotein gene.

DNase I footprinting assays were performed to identify the binding sites for putative trans-acting factors involved in the control of alpha-fetoprotein (AFP) gene expression using mouse AFP promoter fragments (-839 to +56) and nuclear protein extracts from fetal, newborn, and adult livers and from brain and kidney. Our studies have shown that with nuclear protein from adult mouse liver, there are 14 protected regions in the AFP promoter up to -839 base pairs (bp). Region I (-82 to -43) was protected by at least three different factors, one of which is CCAAT-binding/enhancer-binding protein. This region is highly conserved in the mouse, rat, and human AFP genes and has been shown previously to be essential for the regulation of tissue-specific expression in mouse. Differences in DNase I protection with fetal, newborn, and adult nuclear proteins have been observed in the proximal promoter region (up to -202 bp) and in regions further upstream (up to -839 bp). Significant differences among liver, kidney, and brain nuclear protein-binding sites have also been observed. In these studies, we have mapped the fetal and adult nuclear protein-binding sites of the cis-acting DNA sequences of the mouse AFP proximal promoter (up to -200) and have identified specific protein-binding sites in the distal promoter (-200 to -839). We have also identified the sites of the AFP promoter which bind nuclear proteins from highly differentiated tissues in which AFP is not expressed.     

Modulation of interferon gene transcription by positive and negative cellular factors

In vivo competition experiments were designed to identify the role of trans-acting cellular factors in the virus-inducible activation of the interferon-beta promoter. Co-transfection of a constant amount of IFN-beta/CAT test gene and increasing amounts of competitive DNA containing different IFN regulatory domains into human epithelioid 293 cells identified a low abundance, positive cellular factor(s) that interacts with the IFN regulatory region. Competition of the factor decreases virus-induced and constitutive level expression of the IFN-beta promoter, and also partially inhibits expression from the SV40 promoter. Negative regulatory effects produced by factors interacting with the IFN upstream region (-135 to -202) and with the SV40 enhancer were also observed.