A functional NF-kappaB enhancer element in the first intron contributes to the control of c-fos transcription.

Eukaryotic gene transcription is controlled not only by gene promoters but also by intragenic cis-elements. Such regulation is important for the transcription of immediate early genes (IEGs) and in particular for the c-fos gene, the first intron of which contains many potential transcription factor binding elements. In the present study, we addressed the intronic control of c-fos transcription by the NF-kappaB signalling pathway in the neuroendocrine cell line GH4C1. Tumour necrosis factor alpha (TNFalpha) activating the NF-kappaB signalling pathway induced transcription of the c-fos gene and enhanced thyrotropin-releasing hormone-stimulated (TRH-stimulated) c-fos transcription. To examine the effects of NF-kappaB, the presumed NF-kappaB binding sequence in the first intron was mutated or deleted from c-fos reporter gene constructs. When GH4C1 cells transfected with the reporter constructs were stimulated by TNFalpha, the induced expression was significantly diminished. Double-stranded short DNA with the intronic NF-kappaB binding consensus sequence interacted directly with NF-kappaB p50 protein in vitro; mutation of 3 nucleotides destroying the consensus abolished the in vitro interaction. The importance of NF-kappaB for c-fos expression was also supported by RNA interference experiments; knock-down of NF-kappaB p50 suppressed TNFalpha-induced c-fos expression. In addition, chromatin immunoprecipitation indicated that NF-kappaB occupied the first intron of the c-fos gene in vivo. In conclusion, NF-kappaB enhances c-fos transcription via the direct binding to a response element situated in the first intron.