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

Abstract

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-κB signalling pathway in the neuroendocrine cell line GH4C1. Tumour necrosis factor α (TNFα) activating the NF-κB 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-κB, the presumed NF-κB 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 TNFα, the induced expression was significantly diminished. Double-stranded short DNA with the intronic NF-κB binding consensus sequence interacted directly with NF-κB p50 protein in vitro; mutation of 3 nucleotides destroying the consensus abolished the in vitro interaction. The importance of NF-κB for c-fos expression was also supported by RNA interference experiments; knock-down of NF-κB p50 suppressed TNFα-induced c-fos expression. In addition, chromatin immunoprecipitation indicated that NF-κB occupied the first intron of the c-fos gene in vivo. In conclusion, NF-κB enhances c-fos transcription via the direct binding to a response element situated in the first intron.

Introduction

Gene transcription is controlled by transcription factors binding to specific cis-acting elements. These are found both in gene promoters as well as in distal loci and function to silence or enhance gene transcription regardless of their orientation and at a distance from the genes (Lodish et al., 1999). In addition, these cis-acting elements are decisive for the correct timing of gene transcription, for example, to control cell growth and differentiation. According to the cellular status and its environment (e.g. extra-cellular stimuli), specific transcription factors interact with their targeting cis-acting elements to finely tune transcription of specific genes. Such transcription control is particularly important for the transcription of immediate early genes (IEGs).

The translated products of IEGs, which encode mainly transcription factors, are early regulators for cellular responses (e.g. cell growth, differentiation). Transcription of IEGs is activated rapidly and transiently in response to various extra-cellular stimuli without de novo producing of proteins (Hazzalin and Mahadevan, 2002). In contrast, transcription of late response genes is activated following the synthesis of IEG products. Many mechanisms which control the transcription of IEGs (e.g. c-fos, c-myc, heat shock genes) have been well characterized. For instance, transcription of the c-fos gene is controlled by signaling cascades addressing cis-elements in the promoter (e.g. SRE, CRE) (Kovács, 1998, Sng et al., 2004.). In addition to promoter cis-elements, intragenic cis-elements are also known to play key roles for the transcription; the first intron of the c-fos gene includes several regulatory cis-elements which are essential for the induction of its transcription in neuroendocrine cells (Finkbeiner, 2001). Indeed, the deletion or mutation of the intronic region diminishes the transcription activated via intracellular Ca²⁺ signaling pathway in pituitary cells (van Haasteren et al., 2000) and via glucose and gut hormones in pancreatic β-cells (Susini et al., 2000).

Which trans-elements control c-fos transcription via the cis-elements in the first intron? Although a few transcription factors (e.g. DREAM [Carrión et al., 1999], Sp1 [Suske, 1999]) have been thought to interact with their consensus cis-elements situated in the first intron (Finkbeiner, 2001, Susini et al., 2000, van Haasteren et al., 2000), the significance of the interactions remains unclear. In addition to such cis-elements, we found the unique NF-κB binding sequence in the first intron. The NF-κB family consists of RelA (p65), RelB, c-Rel, p50 and p52, and the classical and major dimer form is composed of RelA (p65) and p50 (Baldwin, 1996, Beinke et al., 2004). NF-κB heterodimers function to activate gene transcription whereas p50 homodimers function either as activators or repressors (Baldwin, 1996, Ghosh et al., 2002, Ghosh et al., 1998, Kang et al., 1992, Kurland et al., 2001, Plaksin et al., 1993). The NF-κB pathway is induced by a very large repertory of agents; the prominent among them are pro-inflammatory cytokines, bacterial products, protein synthesis inhibitors, oxidative stress, ultraviolet light and phorbol esters (Baeurle, 1991, Baldwin, 1996, Grilli et al., 1993). As a consequence, NF-κB contributes to the control of the expression of genes important for diverse biological processes (e.g. immune, stress responses, embryo or cell lineage development, cell apoptosis, cell cycle progression, inflammation, oncogenesis) (Chen et al., 2002, Ghosh et al., 2002, Li et al., 2002).

The question we addressed in the present study is whether and how the NF-κB signalling pathway can contribute to the control of c-fos transcription in the neuroendocrine GH4C1 cells. To this end, we tested whether the presumed NF-κB binding sequence functioned as an enhancer element in vivo for c-fos transcription induced by tumour necrosis factor α (TNFα). By using BIAcore analysis and chromatin immunoprecipitation (ChIP) experiments, we furthermore confirmed the in vitro and in vivo interaction of the intronic sequence with NF-κB (p50 protein). We conclude that TNFα stimulation regulates c-fos transcription via the NF-κB signalling pathway, NF-κB directly interacting with the intronic enhancer element.