Regulation of the Interferon-Inducible PKR Kinase Gene: The KCS Element Is a Constitutive Promoter Element That Functions in Concert with the Interferon-Stimulated Response Element

Abstract

The RNA-dependent protein kinase PKR plays important roles in the antiviral and antiproliferative actions of IFN. The IFN-inducible promoter of the human PKR gene contains a 15-bp DNA element designated KCS. The KCS element is located 4 bp upstream of the interferon-stimulated response element (ISRE) and is required for both basal and IFN-inducible transcription. We have examined the effect of insertion mutations between the KCS and the ISRE elements, as well as altered orientation of the KCS element relative to the ISRE element, to assess a possible functional interaction between them. Large insertions (>or=93 bp) between the KCS and ISRE elements significantly reduced both basal and IFN-inducible promoter activity. The function of the KCS element was dependent on the orientation of KCS relative to the ISRE element. Multimerization of the KCS element increased both basal and IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) identified IFN-inducible protein complex formation that required both the KCS and the ISRE DNA element sequences. The novel IFN-inducible protein complexes contained the transcription factor STAT1, as shown by supershift analyses and by their presence in extracts prepared from STAT1 wild-type but not from STAT1-/- null cells. These results, taken together, strongly suggest that the KCS and ISRE elements of the human PKR promoter represent a functional unit.

DNA Damage-binding Proteins and Heterogeneous Nuclear Ribonucleoprotein A1 Function as Constitutive KCS Element Components of the Interferon-inducible RNA-dependent Protein Kinase Promoter

“…Selective binding was verified by competition EMSA, using either the wild-type KCS oligo, the substitution mutant KCS(mt6A), or the mutant KCS(mt9T) as the unlabeled double-stranded DNA competitor at 100-fold excess. The conditions for the protein-DNA binding reaction and electrophoresis using 5% native gels at 4°C were as described (17,22), except that fractions from the purification steps were used in place of the nuclear extract as starting material where indicated.…”

“…Human PKR is encoded by a single copy gene that includes 17 exons spanning 50 kb located on human chromosome 2p (11,12). The increased transcription of the PKR gene in IFN-treated cells above the basal level of expression has been firmly established by Northern blot and nuclear run-on analyses with PKR cDNA probes (13,14), by transient transfection promoter analyses using reporter plasmid constructions (15)(16)(17), and by microarray gene profiling analyses (18 -20).…”

“…One is a consensus 13-bp copy of the IFN-stimulated response element (ISRE) responsible for the inducibility of many different genes, including PKR, by type I IFNs (21). The trimeric transcription factor ISGF3 (STAT1, STAT2, and IRF-9) binds to the ISRE of the PKR promoter in an inducible manner (17). The second element is a novel 15-bp element, positioned upstream of the ISRE, that is required for both basal and interferon-inducible transcription.…”

“…This element, designated KCS for kinase conserved sequence, is exactly conserved between the human and mouse PKR promoters in sequence and position relative to the ISRE (11). In contrast to the inducible binding of the ISGF3 factor to ISRE (17,21), the KCS element mediates constitutive protein binding (11).…”

“…Both basal and IFN-inducible promoter activity are impaired by single substitution mutations within either the 5Ј-or 3Ј-halves of the KCS element, muta-tions that also reduce KBP complex formation (16,22). Multimerization of the KCS element increases both basal and inducible transcription, whereas insertions between the two elements significantly reduce both basal and inducible promoter activity, suggesting that the KCS and ISRE elements of the PKR promoter may represent a functional unit (17). The 5Ј-half of the KCS element includes an Sp1-binding site, and antibody supershift studies identified the Sp1 transcriptional activator as a component of the KBP complex (22).…”