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.

Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter.

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca(2+) responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319-33327].