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A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia
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Flanking sequences for the human intercellular adhesion molecule-1 NF-kappaB response element are necessary for tumor

L L Paxton1, L J Li, V Secor

  • 1Emory Skin Diseases Research Core Center, Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

The Journal of Biological Chemistry
|June 20, 1997
PubMed

Insights

The study reveals that specific flanking sequences around a modified kappaB site are crucial for tumor necrosis factor alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) gene transcription. A consensus kappaB site alone can activate transcription, but flanking sequences are essential for proper transcription factor binding.

Area of Science:

  • Molecular Biology
  • Immunology
  • Genetics

Background:

  • Intercellular adhesion molecule-1 (ICAM-1) expression is regulated by cytokines like tumor necrosis factor alpha (TNF-alpha), playing a key role in inflammation and immune responses.
  • Previous studies indicated that a modified kappaB site in the ICAM-1 5'-flanking region mediates TNF-alpha-induced gene transcription.

Purpose of the Study:

  • To investigate the precise role of the modified kappaB site and its surrounding sequences in TNF-alpha-mediated ICAM-1 gene induction.
  • To determine the necessity of flanking sequences for functional transcription factor binding and activation.

Main Methods:

  • Site-directed mutagenesis of the ICAM-1 5'-flanking region, including the modified kappaB site and adjacent nucleotides.
  • Reporter gene assays to measure transcriptional activity.
  • Electrophoretic mobility shift assays (EMSAs) and UV cross-linking/SDS-PAGE to analyze DNA-protein interactions with Rel proteins (p50 and p65).

Main Results:

  • The modified kappaB site alone is insufficient for TNF-alpha induction; both the kappaB site and specific flanking sequences are required.
  • Mutating the modified kappaB site to a consensus kappaB site enabled TNF-alpha-induced transcription, even without flanking sequences.
  • While p65 binds the minimal response element, both p50 and p65 bind a consensus kappaB site.
  • The ICAM-1 kappaB site, lacking flanking nucleotides, failed to form DNA-protein complexes in EMSA and UV cross-linking assays.

Conclusions:

  • Specific flanking sequences surrounding a kappaB binding site are essential for functional transcription factor binding and transactivation.
  • These flanking sequences are critical for TNF-alpha-mediated induction of ICAM-1 gene expression.

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