Embryonic but Not Postnatal Reexpression of Hepatocyte Nuclear Factor 1α (HNF1α) Can Reactivate the Silent Phenylalanine Hydroxylase Gene in HNF1α-Deficient Hepatocytes

Abstract

The failure to transcribe the phenylalanine hydroxylase (PAH) gene in the liver of hepatocyte nuclear factor 1α (HNF1α)-deficient mice correlated with DNA hypermethylation and the presence of an inactive chromatin structure (M. Pontoglio, D. M. Faust, A. Doyen, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 17:4948–4956, 1997). To evaluate the precise role played by HNF1α, DNA methylation, or histone acetylation in PAH gene silencing, we examined conditions that could restore PAH gene expression in HNF1α-deficient hepatocytes. We show that reactivation of PAH transcription can be achieved by reexpression of HNF1α in embryonic (i.e., embryonic day 12.5 [e12.5] to e13.5) hepatocytes but not in fetal (e17.5), newborn, and adult HNF1α-deficient hepatocytes. This defines a temporal competence window during which HNF1α can act to (re)program PAH gene transcription. We also show that PAH gene silencing can be partially relieved in HNF1α-deficient hepatocytes by treatment with the demethylating agent 5-azacytidine, even in the absence of HNF1α. Treatment using 5-azacytidine combined with trichostatin, a histone deacetylase inhibitor, resulted in a synergistic reactivation of the silenced PAH gene in adult hepatocytes, but this activity was not further increased by HNF1α reexpression. These results suggest that the HNF1α homeoprotein is involved in stage-specific developmental control of the methylation state and chromatin remodeling of the PAH gene.

ACKNOWLEDGMENTS

B.V. was supported by a postdoctoral fellowship from the Association pour la Recherche contre le Cancer (ARC).

We are grateful to T. Bordet, J. E. Guidotti, and A. Mignon for advice on adenovirus production and preparation of mouse hepatocyte primary cultures. We thank D. Faust, M. C. Weiss, C. Cheret, A. Doyen, L. Gresh, and P. Marianneau for fruitful discussions and advice. We also thank J. Weitzman for critical reading of the manuscript.