Constitutive and interleukin-1 (IL-1)-inducible factors interact with the IL-1-responsive element in the IL-6 gene.

The interleukin-6 (IL-6) promoter is rapidly and transiently activated with other cytokines, including IL-1, tumor necrosis factor, and platelet-derived growth factor, as well as phorbol esters and agents that increase intracellular cyclic AMP. In this study, we have investigated cis-acting regulatory elements and trans-acting factors responsible for IL-1-induced IL-6 gene expression. Studies on the 5' deletion mutants of the human IL-6 gene suggested that the IL-1-responsive element was mapped within the IL-6 promoter region (-180 to -123) which was homologous to the c-fos serum-responsive enhancer element. Gel retardation assay identified two types of nuclear factors that bound to this region, one constitutive and the other inducible. These two factors recognized a 14-base-pair (bp) palindromic sequence, ACATTGCACAATCT. Furthermore, three copies of this 14-bp palindrome conferred IL-1 responsiveness to the basal enhancerless IL-6 promoter, indicating that a 14-bp-dyad symmetry sequence was an IL-1-responsive element in the IL-6 gene.     

An element with palindromic structure is required for the expression of TBP (TATA box-binding protein) gene in Drosophila melanogaster

Previously we showed that the 5'-flanking regions between -261 and -207 of the Drosophila melanogaster TBP (TATA box binding protein) gene is important for its expression. We further made serial deletion mutants in this region and analyzed their promoter activities using the transient transfection assay. We found that the 16 bp deletion from -261 to -245 greatly reduces the promoter activity of the Drosophila TBP gene. The 16 bp DNA element contains half of a 11 bp long palindromic sequence, CTTTT-GAAAAG. Disruption of the palindromic sequence by site-directed mutagenesis severely affected promoter activity. In addition, the electrophoretic mobility shift assay showed that the oligonucleotide containing the palindromic sequence can make specific DNA/protein complexes when it was mixed with the Drosophila nuclear extract, suggesting that it interacts with nuclear protein(s). Our data suggest that the palindromic sequence has a critical role in the expression of the Drosophila TBP gene.     

An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.     

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.     

A jasmonate-responsive element within the A. thaliana vsp1 promoter

The vsp1 gene of Arabidopsis thaliana encodes a storage protein that accumulates in vegetative organs. Transgenic plants expressing a vsp1 promoter-gus (beta-glucuronidase) gene fusion were found to contain high GUS activity when challenged with jasmonate, a volatile plant hormone. The induction of vsp1-gus expression by jasmonate could be measured in tobacco mesophyll protoplasts, after transient expression. A number of deletions were operated in the vsp1 promoter in order to locate its jasmonate-responsive element. A 41 bp sequence taken approximately 150 bp upstream of the vsp1 TATA box could confer jasmonate responsiveness upon a short CaMV 35S promoter. Whereas the deletion of a CAAT box-like element within the 41 bp sequence did not affect promoter activity, mutation of a short palindromic motif completely abolished jasmonate responsiveness. This motif shows no homology with the jasmonate-responsive elements of other promoters.     

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.     

Analysis of the Promoter of the Auxin-Inducible Gene, parC, of Tobacco

The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) –226 and –54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt –226 to –151and from nt –84 to –54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt –226 to –151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt –226 to –84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996)     

Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence.

The induction of the viral lytic cycle in latently Epstein-Barr virus (EBV)-infected B cells is initiated by activation of the BZLF1 gene, whose expression is sufficient to disrupt EBV latency, suggesting that BZLF1 acts as the switch to change from a latent to a lytic replicative cycle. In the present studies, a series of deletion plasmids encompassing positions bp -552 to +12 of the BZLF1 promoter were constructed and tested for their response to anti-immunoglobulin (anti-Ig), an inducer of the viral lytic cycle, upon transfection into EBV-negative and -positive lymphoid cells. The promoter consisted of three functionally distinct regions. Region I (bp -552 to -221) had a negative influence on promoter activity; its deletion made the promoter highly responsive to anti-Ig. Region II (bp -203 to -177) was important for conferring responsiveness to anti-Ig. The response to anti-Ig did not require the presence of the EBV genome or EBV gene products. This sequence also enhanced expression of the chloramphenicol acetyltransferase (cat) gene from the simian virus 40 promoter in response to anti-Ig, even when inserted downstream of the cat gene. Region III (-134 to -116) was a positive element that was transactivated by the BZLF1 gene product.