p53-Mediated Repression of Alpha-Fetoprotein Gene Expression by Specific DNA Binding

Abstract

Aberrant expression of the alpha-fetoprotein (AFP) gene is characteristic of a majority of hepatocellular carcinoma cases and serves as a diagnostic tumor-specific marker. By dissecting regulatory mechanisms through electromobility gel shift, transient-transfection, Western blot, and in vitro transcription analyses, we find that AFP gene expression is controlled in part by mutually exclusive binding of two trans-acting factors, p53 and hepatic nuclear factor 3 (HNF-3). HNF-3 protein activates while p53 represses AFP transcription through sequence-specific binding within the previously identified AFP developmental repressor domain. A single mutation within the DNA binding domain of p53 protein or a mutation of the p53 DNA binding element within the AFP developmental repressor eliminates p53-repressive effects in both transient-transfection and cell-free expression systems. Coexpression of p300 histone acetyltransferase, which has been shown to acetylate p53 and increase specific DNA binding, amplifies the p53-mediated repression. Western blot analysis of proteins present in developmentally staged, liver nuclear extracts reveal a one-to-one correlation between activation of p53 protein and repression of AFP during hepatic development. Induction of p53 in response to actinomycin D or hypoxic stress decreases AFP expression. Studies in fibroblast cells lacking HNF-3 further support a model for p53-mediated repression that is both passive through displacement of a tissue-specific activating factor and active in the presence of tissue-specific corepressors. This mechanism for p53-mediated repression of AFP gene expression may be active during hepatic differentiation and lost in the process of tumorigenesis.

ACKNOWLEDGMENTS

We are especially grateful to Jorge Bezerra for invaluable cooperation in the preparation of developmentally staged hepatic extracts, to Ling Sang for expert technical assistance, and to J. Ma, K. Fukasawa, A. Nardulli, and N. Denko for critical comments. We are indebted to G. Lozano, K. Zaret, P. Brindle, B. Spear, and B. Aronow for materials which were essential for these studies.

This work was supported by National Institutes of Health (NIH) grant GM53683 and an American Cancer Society Junior Faculty Research Award to M.C.B. Support to A.J.C. through an NIH National Research Service Award (CA73083) is also gratefully acknowledged.