Putative NF-κB Binding Sites Research Articles

Tumour necrosis factor-α and interferon-γ synergistically activate the RANTES promoter through nuclear factor κB and interferon regulatory factor 1 (IRF-1) transcription factors

Inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically activate expression of the RANTES (regulated upon activation, normal T-cell expressed and secreted) gene, which plays a crucial role in the chemoattraction of leukocytes during the inflammatory response. To understand at the molecular level the mechanism by which the two cytokines activate RANTES gene expression, we determined the requirement of cis-acting elements in the RANTES promoter and trans-acting factors. The murine RANTES promoter contained one putative interferon regulatory factor, IRF, and three putative nuclear factor kappaB (NF-kappaB) binding sites. Specific destruction of the IRF binding site and one of the three NF-kappaB binding sites abolished the inducibility of promoter activity by IFN-gamma and TNF-alpha, respectively. In contrast, mutation of the other two putative NF-kappaB binding sites did not affect RANTES promoter activity significantly. In addition, the RANTES promoter was stimulated by co-transfection of plasmids that expressed either p65, an NF-kappaB family protein, or the IRF-1 transcription factor. RANTES promoters with mutations in the NF-kappaB or IRF binding sites were not stimulated by p65 or IRF-1 expression, respectively. In electrophoretic mobility-shift and immunologic assays, we showed that IRF-1 was induced after cells were treated with IFN-gamma and that NF-kappaB was activated by TNF-alpha treatment. These results demonstrate that both NF-kappaB and IRF-1 transcription factors mediate the induction of RANTES expression via their cognate cis-acting elements when cells are stimulated by TNF-alpha and IFN-gamma.

Cis-acting DNA Elements of Mouse Granulocyte/Macrophage Colony-stimulating Factor Gene Responsive to Oxidized Low Density Lipoprotein

We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5'-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from -97 to -59 and from -59 to -37 and one negative regulatory site from -120 to -97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from -225 to -120 and one negative responsive site from -97 to -59, which contained the NF-kappaB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from -169 to -160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-kappaB binding site. Supershift assay showed that nuclear proteins bound to NF-kappaB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from -169 to -160 was a positive responsive element to Ox-LDL and that the NF-kappaB binding site from -91 to -82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.

Activation of BLV Transcription by NF-κB and Tax

Bovine leukemia virus (BLV) is the causative agent of bovine leukosis, a naturally occurring fatal disease in cattle. BLV transcription is regulated by cellular transcription factors and the virally encoded oncoprotein Tax. In this report, we investigated the functional role of the putative NF-κB binding site recently identified in the BLV promoter. Our studies indicate that the κB binding motif acts as a functional enhancer in the presence of the cellular NF-κB proteins. Furthermore, the κB site together with a single 21-bp repeat confers strong activation of BLV transcription in the presence the NF-κB proteins and Tax. These results suggest that cellular NF-κB may be involved in the regulation of BLV transcription and activation of the virus from latency.

Genomic Organization of the Human ATP1AL1 Gene Encoding a Ouabain-Sensitive H,K-ATPase

The human ATP1AL1 gene belongs to the family of Na,K-ATPase and H,K-ATPase (X,K-ATPases) genes. It encodes a catalytic subunit of hitherto unknown human ouabain-sensitive H,K-ATPase that represents a novel third group of X,K-ATPases distinct from the known Na,K-ATPase and gastric H,K-ATPase. Cloning of the ATP1AL1 gene is described in this report. The exon–intron structure of ATP1AL1 was found to be very similar to that of related genes. It contains 23 exons and spans approximately 32 kb of genomic DNA. All ATP1AL1 exons and 12 of its 22 introns were entirely sequenced. A total of nineAlurepeats were identified in introns. The transcription initiation site was mapped 187 bp upstream of the ATG initiation codon by primer extension and S1nuclease protection analyses of RNA from human skin and colon. Sequence analysis of the 5′-flanking region (1.48 kb) revealed numerous potential binding sites for transcription factors Sp1 and AP2 and one putative NF-κB binding site. The 0.85-kb region from position −484 (5′-flanking region) to position +369 (intron 1) meets the structural criteria of a CpG island. It is suggested that the ATP1AL1 gene contains two poly(A) addition sites that may function in a tissue-specific manner.

An upstream protein interacts with a distinct protein that binds to the cap site of the human interleukin 1β gene

Interleukin 1β (IL-1β) is a proinflammatory cytokine that exhibits a wide variety of biological activities. Genomic sequences that mediate the induction of human IL-1β gene transcription by lipopolysaccharide and phorbol esters are located more than 2,700 bp upstream of the transcriptional start site (cap site). These upstream elements require additional cap site-proximal (CSP) sequences which are necessary for basal transcription of the human IL-1β gene. In addition, these CSP sequences have been shown to mediate both cell type-specific expression of this gene, and trans-activation by some viral proteins. In this study, we report the identification of a novel nuclear protein, termed NFβC, that binds to a DNA sequence which spans the cap site of the human IL-1β gene (positions −12 to +8). We have also identified a second region (positions −305 to −280) containing a putative NF-κB binding site. We show here that this region can bind three distinct nuclear proteins. One protein is similar or identical to NF-κB, a second protein (termed NFβB) binds a distinct sequence that substantially overlaps the 5' half of the NFκB binding sequence, and a third protein (termed NFβD) binds a distinct sequence that substantially overlaps the 3' half of the NFκB binding sequence. Unlike NFκB, NF1βB and NFβD are present in nuclear extracts prepared from unstimulated monocytic cells. Although the NFβD and NFβC binding sequences share no significant similarity, each sequence can specifically compete for the binding of either protein to DNA, whereas oligonucleotides containing only the NFκB or NFβB motifs do not compete for the binding of NFβC or NFβD. This suggests that NFβC and NFβD can specifically interact in vitro, possibly through a common subunit.