A Novel RBP-Jκ-Dependent Switch from C/EBPβ to C/EBPζ at the C/EBP Binding Site on the C-Reactive Protein Promoter

Abstract

Regulation of basal and cytokine (IL-6 and IL-1β)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-κB p50 binding site. In response to IL-6, C/EBPβ and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPζ (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPζ. EMSA using this extract revealed formation of a C/EBPζ-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-Jκ, a transcription factor known to interact with κB sites. RBP-Jκ was required for the formation of C/EBPζ-containing complex. The RBP-Jκ-dependent C/EBPζ-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBPζ abolished both C/EBPβ-induced and (IL-6 + IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPζ occupies the C/EBP site, an action that requires RBP-Jκ. Under induced conditions, C/EBPζ is replaced by C/EBPβ and p50. We conclude that the switch between C/EBPβ and C/EBPζ participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-Jκ into C/EBPζ complexes solely to support the binding of C/EBPζ to the C/EBP site.