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A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

ABSTRACT: The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promoter in human herpesvirus 6-infected cells. Mutational analysis identified a 21-bp element (the EA site) which is located at -73 to -52 bp relative to the transcription start site and is essential for promoter activity. The ability of the EA site to stimulate transcription optimally appears to be strictly dependent upon its distance from the p41 basal promoter. The EA site contains three overlapping sequences, a CAAT-enhancer-binding protein (C/EBP) transcription factor recognition site and two repeat elements. Mobility shift assays using the EA site identified four binding activities (C1 to C4). C1 and C2 are present in both uninfected and infected cells and do not contain C/EBP factors. In infected cells, point mutation of the EA site abrogates C1 and C2 binding activities and destroys transcriptional activity of the p41 promoter. C3 and C4 are present in uninfected cells only and were found to contain C/EBP factors. These findings indicate that in infected cells, transcriptional stimulation of the p41 promoter by the EA site requires C1 and C2 binding activities. These results further suggest that transcriptional activity may also depend upon the elimination of C3 and C4 binding activities.

SUBMITTER: Thompson JR

PROVIDER: S-EPMC236373 | biostudies-other | 1994 Jul

REPOSITORIES: biostudies-other

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A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

Thompson J R JR Agulnick A D AD Ricciardi R P RP

Journal of virology 19940701 7

The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promo ...[more]

PMID: 8207821

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Project description:Trichoderma reesei can produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of the cbh1 promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in the xyn1 promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One such cis element, an inverted repeat, plays a crucial role in the inducibility of the xyn1 promoter. We investigated the impact of the properties of such cis elements by shuffling them by insertion, exchange, deletion, and rearrangement of cis elements in both the cbh1 and xyn1 promoter. A promoter-reporter assay using the Aspergillus nigergoxA gene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase in cbh1 promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances of cis elements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances of cis elements have large impacts on the strength of the cbh1 promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically important cbh1 promoter can be improved by cis element rearrangement.IMPORTANCE In the present study, we demonstrate that the arrangement of cis elements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances of cis elements to the transcription start site. Furthermore, we found that the configuration of cis elements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement of cis elements is an important factor that should not be overlooked when enhancement of gene expression is desired.

Project description:The observation that human herpesvirus 6 (HHV-6) can induce CD4 gene transcription and expression in CD4(-) cells was reported several years ago (P. Lusso, A. De Maria, M. Malnati, F. Lori, S. E. DeRocco, M. Baseler, and R. C. Gallo, Nature 349:533-535, 1991) and subsequently confirmed (P. Lusso, M. S. Malnati, A. Garzino-Demo, R. W. Crowley, E. O. Long, and R. C. Gallo, Nature 362:458-462, 1993; G. Furlini, M. Vignoli, E. Ramazzotti, M. C. Re, G. Visani, and M. LaPlaca, Blood 87:4737-4745, 1996). Our objective was to identify the mechanisms underlying such phenomena. Using reporter gene constructs driven by the CD4 promoter, we report that HHV-6 can efficiently transactivate such genetic elements. Activation of the CD4 promoter occurs in the presence of the viral DNA polymerase inhibitor phosphonoformic acid, which limits expression to the immediate-early and early classes of viral genes. Using deletion mutants and specific CD4 promoter mutants, we identified an ATF/CRE binding site located at nucleotides -67 to -60 upstream of the CD4 gene transcription start site that is important for HHV-6 transactivation. The ATF/CRE site is also essential for CD4 promoter activation by forskolin, an activator of adenylate cyclase. Using electrophoretic mobility shift assays and specific antibodies, we showed that CREB-1 binds specifically to the -79 to -52 region of the CD4 promoter. Last, we have identified two open reading frames (ORFs) of HHV-6, U86 and U89 from the immediate-early locus A, that can transactivate the CD4 promoter in HeLa cells. However, transactivation of the CD4 promoter by ORFs U86 and U89 is independent of the CRE element, suggesting that additional HHV-6 ORFs are likely to contribute to CD4 gene activation. Taken together, our results will help to understand the complex interactions occurring between HHV-6 and the CD4 promoter and provide additional information regarding the class of transcription factors involved in the control of CD4 gene expression.

Dataset Information

A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

Publications

A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

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