Interferon-induced transcription of a gene encoding a 15-kDa protein depends on an upstream enhancer element.

Abstract

A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the "CCAAT box." Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene.Many polypeptide hormones, such as the interferons, exert their effect by binding to plasma membrane receptors and, subsequently, affecting the transcriptional activity of a specific set of genes (1-5). The mechanism by which this cell surface signal is transduced into the nucleus, causing an alteration of gene expression, remains an active area of investigation. We are studying this transduction by analyzing transcriptional control elements that respond to interferon treatment.In this report we describe the genomic cloning and functional characterization of an interferon-sensitive transcription unit encoding a 15-kDa protein (6). Interferon treatment results in a transient stimulation of transcriptional activity of the gene, as has been demonstrated for other interferonsensitive genes (4, 7). We therefore refer to the group as ISG (interferon-stimulated gene) and to this member as ISGCS.Characterization of the interferon-responsive DNA element of ISGI5 was performed by DNA transfection assays using recombinant hybrid clones. To distinguish between expression of the transfected and the endogenous gene, we made recombinants that contained the upstream region ofISG15 (to nucleotide +44) fused with the 3' region of the adenovirus EJB gene. Deletions from the 5' sequences of the basic construct revealed that the region from -115 to -% (relative to the transcription initiation site) was required for interferon stimulation of the promoter. Comparison of this DNA sequence to the region also known to confer interferonsensitivity to another gene (ISG54, ref. 8) showed a conserved oligonucleotide stretch. In each case the conserved region in the two genes is near the "CAAT box" but is in the opposite orientation in the two genes.This regulatory...

Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity

“…2A). ISG54 and ISG15 transcription increased substantially in response to IFN, whereas actin transcription remained approximately uniform, as expected (18). However, addition of TSA together with IFN impaired transcriptional induction of ISG54 and ISG15 by 60% and 75%, respectively, while having no effect on actin transcription.…”

“…Run-on transcription experiments (15) used Ϸ10 million cells per point. DNA probes were the following: Vector (pGEM1, Promega), ␥-actin cDNA (16), ISG54 EcoRI-TaqI fragment from exon 2 (17), and ISG15 TaqI fragment from exon 2 (18). After hybridization, filter signals were quantified by PhosphorImager (Bio-Rad).…”

Regulatory Effects of Mammalian Target of Rapamycin-activated Pathways in Type I and II Interferon Signaling

“…For instance, it is well established that the promoter of the Isg15 gene contains three ISRE elements and that the STAT pathway regulates its transcriptional activation (50). In fact, the original definition of ISREs was based on their identification and description of their presence in the Isg15 gene promoter (50,51). Our findings now provide direct evidence that translational control of the Isg15 gene product is regulated by cap-dependent translation, as evidenced by the enhanced up-regulation of IFN␣-dependent ISG15 protein expression in cells with targeted disruption of the 4e-bp1 gene.…”

A Novel Type I Cytokine Receptor Is Expressed on Monocytes, Signals Proliferation, and Activates STAT-3 and STAT-5

“…4, A and B). To exclude the presence of interferon-stimulated gene factor 3, a complex containing activated STAT-1 and STAT-2, we also tested our extracts on the interferon-stimulated response element (ISRE) (25) probe, but did nor observe any hGH-specific gelshifts (not shown). Thus, no STAT molecules other than STAT-3 and STAT-5 are activated by hGH-R⅐GLM-R under these conditions.…”