Measurement of NF-κB transcriptional activity and identification of NF-κB cis-regulatory elements using luciferase assays.
The NF-κB family of transcription factors is activated in response to numerous environmental stimuli and coordinates the transcriptional response to immunoreceptors such as the Toll-like receptors, cytokine receptors, and antigen receptors, growth factors, survival factors, and stress signals such as ultraviolet irradiation and oxidative stress. The transcriptional targets of these various pathways include approximately 500 experimentally indentified genes, and it is highly likely that many others remain to be discovered. A genome-wide analysis of NF-κB-chromatin interactions has revealed a surprisingly large number of NF-κB binding sites across the entire genome, many of which are found in intergenic regions and many more do not appear to be associated with changes in transcription of nearby genes. Assessing the consequences of NF-κB binding at genomic sites is therefore essential to determine the functional role of NF-κB in regulating the expression of specific genes. Luciferase-based reporter assays provide a robust and flexible method to test the contribution of specific NF-κB sites to the regulation of gene transcription. The methods described in this chapter may be applied to any promoter sequence and used in a variety of cell lines and conditions to provide critical information on the regulation of gene expression by NF-κB.
Institute of Infection, Immunology and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow, G12 8TA, UK.
Patricia E
PE
Collins
Christine
C
O'Carroll
Ruaidhrí J
RJ
Carmody
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D015533:Transcriptional Activation; D014162:Transfection; D014169:Transformation, Bacterial
D016328:NF-kappa B; D008156:Luciferases