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Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element
R Hooft van Huijsduijnen1, R Pescini, J F DeLamarter
1GLAXO Insitute for Molecular Biology, Geneva, Switzerland.
Nucleic Acids Research
|August 11, 1993
Summary
Researchers identified two distinct nuclear factor-kappa B (NF-κB) complexes binding the E-selectin promoter. One complex contains p50/p65, while the other includes p50/p75, influenced by DNA sequence length.
Area of Science:
- Molecular Biology
- Cellular Biology
- Immunology
Background:
- E-selectin plays a crucial role in immune cell trafficking.
- Nuclear factor-kappa B (NF-κB) is a key transcription factor regulating E-selectin expression.
- Understanding NF-κB binding dynamics is vital for controlling inflammatory responses.
Purpose of the Study:
- To investigate proteins binding the E-selectin promoter NF-κB element in its natural DNA context.
- To characterize the composition of different NF-κB complexes.
- To determine factors influencing the formation of these complexes.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) using nuclear extracts from cytokine-induced cells.
- Subunit-specific antisera to block complex formation.
- DNA-protein cross-linking experiments.
- Probes extending beyond the NF-κB recognition site.
Main Results:
- Two distinct NF-κB complexes were detected binding the E-selectin promoter.
- The faster migrating complex comprised NF-κB p50 and p65 subunits.
- The slower migrating complex consisted of p50 and a p65-related protein, p75.
- Complex formation was dependent on DNA sequence length downstream of the NF-κB site.
Conclusions:
- The presence of extended DNA sequences ( >5 base pairs) downstream of the NF-κB site is crucial for the formation of the larger p50/p75 complex.
- An intact NF-κB binding site is necessary for the larger complex, potentially requiring activated p50.
- The p75-containing complex may have a regulatory role on the E-selectin promoter.