trans activation of type 1 interferon promoters by simian virus 40 T antigen.

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.     

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.     

UV inducibility of rat proliferating cell nuclear antigen gene promoter.

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase delta, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth-regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose-dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides -70 and +125, which contains an AP-1 site and a downstream proximal ATF/CRE site. While mutation of the AP-1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent.     

Building a metal-responsive promoter with synthetic regulatory elements.

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.     

cis-regulatory elements involved in ultraviolet light regulation and plant defense.

An elicitor-regulated transient expression system was established in soybean protoplasts that allowed the identification of cis-regulatory elements involved in plant defense. The 5' region of an ultraviolet (UV) light-inducible and elicitor-inducible chs gene (chs1) of soybean was subjected to deletion analysis with the help of chimeric chs-nptII/gus gene constructs. This analysis delimited the sequences necessary for elicitor inducibility to -175 and -134 of the chs1 promoter. The same soybean sequences were able to direct elicitor inducibility in parsley protoplasts, suggesting a conserved function of cis-acting elements involved in plant defense. In addition, this region of the soybean promoter also promotes UV light inducibility in parsley protoplasts. However, in contrast to the elicitor induction, correct regulation was not observed after UV light induction when sequences downstream of -75 were replaced by a heterologous minimal promoter. This result indicates that at least two cis-acting elements are involved in UV light induction.     

Repression of the murine interferon alpha 11 gene: identification of negatively acting sequences.

The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.     

Identification of two distinct nuclear factors with DNA binding activity within the glucocorticoid regulatory region of the rat alpha-1-acid glycoprotein promoter

Efficient glucocorticoid induction of alpha-1-acid glycoprotein (AGP) mRNA in rat hepatoma cells HTC (JZ-1) requires the activity of one or more preexisting and labile proteins acting cooperatively with the glucocorticoid receptor. Inhibiting protein synthesis markedly diminishes the glucocorticoid induction of rat AGP mRNA without affecting the inducibility of other glucocorticoid inducible genes such as the mouse mammary tumor virus (MMTV) or tyrosine amino transferase (TAT). The sequences responsible for conferring glucocorticoid inducibility to the rat AGP gene have localized on the AGP promoter between nucleotides -121 and -42. A typical glucocorticoid regulatory element (GRE) is found between residues -121 and -105 and downstream of this are the sequences (-90 to -42) responsible for the cycloheximide inhibition of the hormonal induction (10). Using mobility shift assays we have characterized the binding of two proteins or complexes of proteins to this promoter region (-90 to -64). Our data show that the binding of these factors (called ANF-1 and ANF-2) to the DNA is highly specific, and is not directly affected by cycloheximide. Furthermore a second binding site for ANF-2 has been localized in the AGP regulatory region to a sequence that overlaps the GRE.