Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites—the P2 site—located between –173 and –150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes—a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at –173 to –150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.© 1997 Elsevier Science B.V. All rights reserved.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

AP-1/jun binding sites mediate serum inducibility of the human vimentin promoter.

The vimentin gene is inducible by serum in quiescent Balb/c 3T3 cells, but the molecular mechanism of this induction is unknown. The results presented here represent a first step towards the elucidation of the pathway of events leading from growth factor-receptor interaction to this induction. A series of Bal 31 deletions of the human vimentin promoter are used to show that a sequence residing at -700 is responsible for the serum, and also TPA inducibility of this gene. This sequence is able to confer serum inducibility upon uninducible constructs regardless of its position and orientation, indicating that it is this element alone which is required for this induction. The isolated sequence is a strong enhancer as well. Further deletions and the use of synthetic oligonucleotides demonstrate that a 24-mer containing two AP-1/jun binding sites confer both serum and TPA inducibility upon the human vimentin gene. Gel retardation analysis confirms that this sequence binds an AP-1 -like protein.