Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss)

The upstream flanking region of the rainbow trout (Oncorhynchus mykiss) IL-1beta1 gene has been cloned and characterised functionally using luciferase-based reporter gene constructs, and the transcription start site (TSS) confirmed by RLM-RACE. A TATA box was present 27 bp upstream of the TSS, with an NF-kappaB site 19 bp upstream of the TATA box. Within 1217 bp of upstream sequence, 3 sites for NF-kappaB, 10 sites for NF-IL6, 15 sites for AP1, 6 sites for AN, 2 sites for CHOP/CEBPalpha and I site for SP1 and PU.1 were identified. Seven potential sites for the transcription repressor Gfi-1 were also identified. Analysis of eight IL-1beta1s promoter luciferase constructs transfected into a trout fibroblast (RTG-2) cell line known to constitutively express IL-1beta revealed that in the absence of intron 1, very low luciferase activity was detectable. All of the constructs containing intron 1 gave clear luciferase activity, with the highest luciferase activity detected with construct P2-4 containing 617 bp of upstream sequence. As little as 82 bp of upstream sequence gave relatively strong luciferase activity, a region containing both a PU.1 and NF-kappaB site. That NF-kappaB is a transcription factor required for expression of the trout IL-1beta1 gene was confirmed using inhibitor studies with lipopolysaccharide (LPS)-stimulated macrophages. Both trout recombinant IL-1beta and LPS were able to increase luciferase activity in the reporter constructs, especially in those containing the most upstream sequence with the lowest constitutive expression. The possibility that an upstream repressor is functioning to inhibit constitutive expression of IL-1beta in this species is discussed. (C) 2002 Elsevier Science B.V. All rights reserved.