Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter.

Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C.     

C/EBP alpha and Ets protein family members regulate the human myeloid IgA Fc receptor (Fc alpha R,CD89) promoter

Fc alpha R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc alpha R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.     

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.     

Regulation of IL-12 p40 promoter activity in primary human monocytes: roles of NF-kappaB, CCAAT/enhancer-binding protein beta, and PU.1 and identification of a novel repressor element (GA-12) that responds to IL-4 and prostaglandin E(2)

Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14(+) monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-kappaB, and ETS were only found upon stimulation with LPS and IFN-gamma. In contrast, a footprint over a purine-rich sequence at -155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE(2). Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.     

Sp1 represses IL-2 receptor alpha chain gene expression

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.