A novel RBP-Jκ-dependent switch from C/EBPβ to C/EBPξ at the C/EBP binding site on the C-reactive protein promoter

Regulation of basal and cytokine (IL-6 and IL-1 beta)-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-kappa B p50 binding site. In response to IL-6, C/EBP beta 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/EBP beta. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBP beta. Such treated nuclei contain only C/EBP zeta (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBP zeta. EMSA using this extract revealed formation of a C/EBP zeta containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-JK, a transcription factor known to interact with kappa B sites. RBP-J kappa was required for the formation of C/EBP zeta-containing complex. The RBP-J kappa-dependent C/EBP zeta-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/EBP zeta abolished both C/EBP beta-induced and (IL-6 + IL-1 beta)induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBP zeta occupies the C/EBP site, an action that requires RBP-J kappa. Under induced conditions, C/EBP zeta is replaced by C/EBP beta and p50. We conclude that the switch between C/EBP beta and C/EBP zeta participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-J kappa into C/EBP zeta complexes solely to support the binding of C/EBP zeta to the C/EBP site.