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

Tiehui Wang, Jun Zou, Charles Cunningham, Christopher J Secombes

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25 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)108-116
Number of pages9
JournalBiochimica et Biophysica Acta (BBA) - Gene Structure and Expression
Volume1575
Issue number1-3
Early online date2 Apr 2002
DOIs
Publication statusPublished - 3 May 2002

Keywords

  • rainbow trout
  • interleukin-1 beta
  • promoter
  • transcription start site
  • transcription factors
  • repressor
  • NF-kappa-B
  • necrosis-factor-alpha
  • protein-kinase-C
  • gene-expression
  • transcriptional regulation
  • molecular-cloning
  • IL-1-beta gene
  • plasmid DNA
  • cell-line
  • activation
  • 5' flanking region
  • animals
  • base sequence
  • cloning
  • molecular
  • gene expression regulation
  • interleukin-1
  • molecular sequence data
  • Oncorhynchus mykiss
  • promoter regions
  • sequence analysis
  • DNA

Cite this

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title = "Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss)",
abstract = "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.",
keywords = "rainbow trout, interleukin-1 beta, promoter, transcription start site, transcription factors, repressor, NF-kappa-B, necrosis-factor-alpha, protein-kinase-C, gene-expression, transcriptional regulation, molecular-cloning, IL-1-beta gene, plasmid DNA, cell-line, activation, 5' flanking region , animals, base sequence, cloning, molecular, gene expression regulation, interleukin-1, molecular sequence data , Oncorhynchus mykiss, promoter regions, sequence analysis, DNA",
author = "Tiehui Wang and Jun Zou and Charles Cunningham and Secombes, {Christopher J}",
year = "2002",
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doi = "10.1016/S0167-4781(02)00235-X",
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TY - JOUR

T1 - Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss)

AU - Wang, Tiehui

AU - Zou, Jun

AU - Cunningham, Charles

AU - Secombes, Christopher J

PY - 2002/5/3

Y1 - 2002/5/3

N2 - 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.

AB - 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.

KW - rainbow trout

KW - interleukin-1 beta

KW - promoter

KW - transcription start site

KW - transcription factors

KW - repressor

KW - NF-kappa-B

KW - necrosis-factor-alpha

KW - protein-kinase-C

KW - gene-expression

KW - transcriptional regulation

KW - molecular-cloning

KW - IL-1-beta gene

KW - plasmid DNA

KW - cell-line

KW - activation

KW - 5' flanking region

KW - animals

KW - base sequence

KW - cloning

KW - molecular

KW - gene expression regulation

KW - interleukin-1

KW - molecular sequence data

KW - Oncorhynchus mykiss

KW - promoter regions

KW - sequence analysis

KW - DNA

U2 - 10.1016/S0167-4781(02)00235-X

DO - 10.1016/S0167-4781(02)00235-X

M3 - Article

C2 - 12020825

VL - 1575

SP - 108

EP - 116

JO - Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

JF - Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

SN - 0167-4781

IS - 1-3

ER -