Functional analysis of multiple promoters of the rat insulin-like factor II gene

We have characterized the multiple promoters of the rat insulin-like growth factor II (rIGFII) gene by in vivo transient expression assay using a series of deletion mutant templates. Among the four promoters (P1, P2, P3 and P6), two (P2 and P3) showed relatively strong promoter activities compared with the other two. One of the four promoters, P2, was further characterized by gel band-shift and footprinting analysis using HeLa cell nuclear extract, showing two retarded bands and at least one protected sequence stretch. The results indicated that P2 has a very simple structure like P3, and consists of no more than 141 base-pairs (bp) including a TATA box and two GC core hexanucleotides. Promoter strength shown by in vivo transient expression in different cell types failed to explain the differential employment of P2 and P3 in these cells, suggesting the involvement of other regulatory mechanisms that might operate only in the native state.     

The human IGF-I gene contains two cell type-specifically regulated promoters.

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

Cell-specific in vivo DNA-protein interactions at the proximal promoters of the pro alpha 1(I) and the pro alpha2(I) collagen genes.

We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proalpha1(I) collagen promoter and the 350 bp mouse proximal proalpha2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proalpha1(I) and proalpha2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proalpha1(I) and proalpha2(I) collagen promoters in vitro , it is only in fibroblasts that these proteins bind to their cognate sites in vivo . We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.     

Comparison of promoter activity in Aleutian mink disease parvovirus, minute virus of mice, and canine parvovirus: possible role of weak promoters in the pathogenesis of Aleutian mink disease parvovirus infection.

Aleutian mink disease parvovirus (ADV) infection causes both acute and chronic disease in mink, and we have previously shown that it is the level of viral gene expression that determines the disease pattern. To study the gene regulation of ADV, we have cloned the P3 ADV and P36 ADV promoters in front of a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and analyzed these constructs by transient transfection in a feline kidney cell line and mouse NIH 3T3 cells. The genes for ADV structural proteins (VP1 and VP2) and the nonstructural proteins (NS-1, NS-2, and NS-3) were cloned into a eukaryotic expression vector, and their functions in regulation of the P3 ADV and P36 ADV promoters were examined in cotransfection experiments. The ADV NS-1 protein was able to transactivate the P36 ADV promoter and, to a lesser degree, the P3 ADV promoter. Constitutive activities of the P3 ADV and P36 ADV promoters were weaker than those of the corresponding promoters from the prototypic parvovirus minute virus of mice (MVM) and canine parvovirus (CPV). Also, the level of transactivation of the P36 ADV promoter was much lower than those of the corresponding P38 MVM and P38 CPV promoters transactivated with MVM NS-1. Moreover, the ADV NS-1 gene product could transactivate the P38 MVM promoter to higher levels than it could transactivate the P36 ADV promoter, while the P36 ADV promoter could be transactivated by MVM NS-1 and ADV NS-1 to similar levels. Taken together, these data indicated that cis-acting sequences in the P36 ADV promoter play a major role in determining the low level of transactivation observed. The P3 ADV and P4 MVM promoters could be transactivated to some degree by their respective NS-1 gene products. However, in contrast to the situation for the late promoters, switching NS-1 proteins between the two viruses was not possible. This finding may indicate a different mechanism of transactivation of the early promoters (P3 ADV and P4 MVM) compared with the late (P36 ADV and P38 MVM) promoters. In summary, the constitutive levels of expression from the ADV promoters are weaker than the levels from the corresponding promoters of MVM and CPV. Moreover, the level of NS-1-mediated transactivation of the late ADV promoter is impaired compared with the level of transactivation of the late promoters of MVM and CPV.(ABSTRACT TRUNCATED AT 400 WORDS)