Regulatory Mechanisms Controlling Human Hepatocyte Nuclear Factor 4α Gene Expression

Abstract

Hepatocyte nuclear factor 4α (HNF-4α) (nuclear receptor 2A1) is an essential regulator of hepatocyte differentiation and function. Genetic and molecular evidence suggests that the tissue-restricted expression of HNF-4α is regulated mainly at the transcriptional level. As a step toward understanding the molecular mechanism involved in the transcriptional regulation of the human HNF-4α gene, we cloned and analyzed a 12.1-kb fragment of its upstream region. Major DNase I-hypersensitive sites were found at the proximal promoter, the first intron, and the more-upstream region comprising kb −6.5, −8.0, and −8.8. By the use of reporter constructs, we found that the proximal-promoter region was sufficient to drive high levels of hepatocyte-specific transcription in transient-transfection assays. DNase I footprint analysis and electrophoretic mobility shift experiments revealed binding sites for HNF-1α and -β, Sp-1, GATA-6, and HNF-6. High levels of HNF-4α promoter activity were dependent on the synergism between either HNF-1α and HNF-6 or HNF-1β and GATA-6, which implies that at least two alternative mechanisms may activate HNF-4α gene transcription. Chromatin immunoprecipitation experiments with human hepatoma cells showed stable association of HNF-1α, HNF-6, Sp-1, and COUP-TFII with the promoter. The last factor acts as a repressor via binding to a newly identified direct repeat 1 (DR-1) sequence of the human promoter, which is absent in the mouse homologue. We present evidence that this sequence is a bona fide retinoic acid response element and that HNF-4α expression is upregulated in vivo upon retinoic acid signaling.

ACKNOWLEDGMENTS

We are indebted to A. Aranda, S. Cereghini, P. Chambon, R. Cortese, R. Costa, T. Evans, F. Lemaigre, D. Mangelsdorf, and M. Parker for providing the indicated reagents. We are grateful to N. Katrakili for expert technical assistance, C. Mamalaki for advice with HS analysis, E. Soutoglou and K. Boulias for helpful discussions, G. Keszler for critical reading of the manuscript, and an anonymous reviewer for helpful comments.

This work was supported by funds from the Greek General Secretariat for Science and Technology, by HFSP RGP-0024, and by EU HPRN-CT-2000-00087 programs.