A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Interferon-responsive regulatory elements in the promoter of the human 2'',5''-oligo(A) synthetase gene.

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.     

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.     

The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site.

The 82-kDa IE2 protein of human cytomegalovirus (HCMV) acts as both a powerful nonspecific trans activator of heterologous promoters and a negative autoregulator of HCMV immediate-early gene expression in transient assays. We show here that the highly specific down-regulation effect occurs in permissive diploid human fibroblast cells as well as in nonpermissive Vero cells and that the target sequences are conserved within the major immediate-early promoters of both HCMV and simian cytomegalovirus. The response sequences were localized between -67 and +30 in the simian cytomegalovirus IE94 promoter and upstream of position +9 in the HCMV IE68 promoter. Deletion of sequences downstream of -14 in a target IE68-CAT gene abolished the negative phenotype and resulted in a reporter gene that was stimulated instead of inhibited by cotransfection with IE2 effector DNA. Insertion of an oligonucleotide containing sequences from between -17 and +9 into the IE68-CAT deletion construction restored autoregulation in either orientation. Furthermore, this same oligonucleotide transferred the full down-regulation phenotype when inserted at +10 into the nonresponsive IE175 promoter from herpes simplex virus. Therefore, a specific response signal that acts at the DNA level must lie within these boundaries. Additional analysis with inserted oligonucleotides containing deletions or point mutations revealed that essential components of the signal lie between positions -12 and +5. Therefore, negative autoregulation by HCMV IE2 in DNA cotransfection systems resembles that for simian virus 40 large T antigen and herpes simplex virus IE175 by acting through a signal located near the cap site, but the target sequence itself bears no resemblance to those utilized in these other viral systems.