P53 regulates CCAAT/Enhancer binding protein β gene expression

Biao Hu; Tianju Liu; Zhe Wu; Sem H Phan

doi:10.1016/j.gene.2023.147675

P53 regulates CCAAT/Enhancer binding protein β gene expression

Gene. 2023 Oct 30:884:147675. doi: 10.1016/j.gene.2023.147675. Epub 2023 Aug 2.

Authors

Biao Hu¹, Tianju Liu², Zhe Wu², Sem H Phan³

Affiliations

¹ Department of Internal Medicine, University of Michigan Medical School, 1600 Huron Parkway, Ann Arbor, MI 48109 USA.
² Department of Pathology, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI 48109 USA.
³ Department of Pathology, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI 48109 USA. Electronic address: shphan@med.umich.edu.

PMID: 37541559
DOI: 10.1016/j.gene.2023.147675

Abstract

Background: The 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.

Methods: In 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.

Results: The 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.

Conclusion: Analysis 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.

Keywords: CCAAT/Enhancer Binding Protein β; P53; Transcriptional regulation; Translational regulation; eIF4e.

MeSH terms

5' Untranslated Regions / genetics
Animals
CCAAT-Enhancer-Binding Protein-beta / genetics
CCAAT-Enhancer-Binding Protein-beta / metabolism
DNA / metabolism
Eukaryotic Initiation Factor-4E* / genetics
Eukaryotic Initiation Factor-4E* / metabolism
Gene Expression
Gene Expression Regulation
Protein Binding
Protein Isoforms / metabolism
Rats
Tumor Suppressor Protein p53* / genetics
Tumor Suppressor Protein p53* / metabolism

Substances

Tumor Suppressor Protein p53
Eukaryotic Initiation Factor-4E
5' Untranslated Regions
CCAAT-Enhancer-Binding Protein-beta
Protein Isoforms
DNA