P53 regulates CCAAT/Enhancer binding protein β gene expression

Abstract

BackgroundThe transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is implicated in diverse processes and diseases. Its two isoforms, namely liver-enriched activator protein (LAP) and liver-enriched inhibitor protein (LIP) are translated from the same mRNA. They share the same C-terminal DNA binding domain except LAP has an extra N-terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, LIP can inhibit LAP transcriptional activity even at substoichiometric levels. However, the regulatory mechanism of C/EBPβ gene expression and the LAP: LIP ratio is unclear. MethodsIn this study, the C/EBPβ promoter sequence was scanned for conserved P53 response element (P53RE), and binding of P53 to the C/EBPβ promoter was tested by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation assay. P53 over-expression and dominant negative P53 expression plasmids were transfected into rat lung fibroblasts and tested for C/EBPβ gene transcription and expression. Western blot analysis was used to test the regulation of C/EBPβ LAP and LIP isoforms. Constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region were used to test the importance of 5′UTR in the control of C/EBPβ LAP and LIP translation. ResultsThe C/EBPβ promoter sequence was found to contain a conserved P53 response element (P53RE), which binds P53 as demonstrated by Electrophoresis Mobility Shift Assay and chromatin immunoprecipitation assays. P53 over-expression suppressed while dominant negative P53 stimulated C/EBPβ gene transcription and expression. Western blot analysis showed that P53 differentially regulated the translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E-BP1. Further studies with constructs containing the LAP 5′untranslated region (5′UTR) or the LIP 5′UTR region showed that the 5′UTR is important in differential control of C/EBPβ LAP and LIP translation. ConclusionAnalysis of the effects of P53 on C/EBPβ expression revealed a novel mechanism by which P53 could antagonize the effects of C/EBPβ on its target gene expression. For the first time, P53 is shown to be a repressor of C/EBPβ gene expression at both transcriptional and translational levels, with a differential effect in the magnitude of the effect on LAP vs. LIP isoforms.