Role of hepatocyte nuclear factor 3γ in the expression of human CYP2C genes

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Abstract

Hepatocyte nuclear factor 3γ (HNF-3γ) is an important transcription factor for the maintenance of specific liver functions. However, its relevance in the expression of human cytochrome P450 (CYP) genes has not yet been explored. Several HNF3 putative binding sites can be identified in human CYP2C 5-flanking regions. Gene reporter experiments with proximal promoters revealed that HNF-3γ transactivated CYP2C8, CYP2C9, and CYP2C19 (25-, 4-, and 4-fold, respectively), but it did not transactivate CYP2C18. However, overexpression of HNF-3γ in hepatoma cells by means of a recombinant adenovirus induced CYP2C9, CYP2C18, and CYP2C19 mRNA (4.5-, 20-, and 50-fold, respectively) but did not activate endogenous CYP2C8. The lack of effect of HNF-3γ on endogenous CYP2C8 could be reversed by treating cells with the deacetylase inhibitor, trichostatin A, suggesting the existence of chromatin condensation around functional HNF3 elements in this gene. We conclude that HNF3γ is an important transcription factor for the hepatic-specific expression of human CYP2C genes. Our results also evidence that efficient transfection tools, such as adenoviral vectors, may be decisive for assessing the role of transcription factor on chromatin organized genes.

Section snippets

Cloning and plasmids construction

The CYP2C promoter–luciferase fusion genes (2C-LUC)—pGL3-2C8, pGL3-2C9, pGL3-2C18, and pGL3-2C19—were obtained by cloning CYP2C8 (1797 bp), CYP2C9 (1836 bp), CYP2C18 (1256 bp), and CYP2C19 (1627 bp) promoters into the enhancerless, promoterless pGL3-Basic vector (Promega, Madison, WI). CYP2C8, CYP2C9, and CYP2C18 promoters were PCR-cloned using the Human GenomeWalker Kit (Clontech, Palo Alto, CA); the PCR-primers used are described in Table 1. To facilitate the posterior cloning of the PCR

Promoter regions of the CYP2C genes contain putative binding sites for HNF-3

CYP2C proximal promoter sequences were analyzed using the MatInspector software [30] and Transfac database [31]. Several HNF-3 motifs were found in CYP2C8, CYP2C9, CYP2C18, and CYP2C19 (Fig. 1A) (conditions for searching were 100% core and 90% matrix similarities). Multiple alignment of CYP2C promoters revealed a certain degree of similarity between CYP2C9 and CYP2C19 promoters, while CYP2C8 and CYP2C18 promoters were more divergent. Basal promoter activities were assayed in HepG2 (hepatic) and

Discussion

Liver is the main organ in which CYP2C genes are expressed [11], [12], [32]. However, hepatoma cells, among them HepG2, have no CYP2C catalytic activities and their mRNAs are hardly detected ([38] and Fig. 5). The mechanism leading to the arrest of such hepatic-specific genes has been related to the loss of tissue-specific enriched transcription factors [26] such as HNF-1α or HNF-3γ, referred to as “hepatic establishment transcriptional factors” [39]. Taking together the relevant role of HNF-3γ

Acknowledgements

We thank C. Corchero and E. Belenchon for their expert technical assistance. This research was supported in part by FEDER Research contract, 1FD97-1496, and FIS Grants 00/1037 and 00/1038.

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    Present address: Cell and Developmental Biology, Fox Chase Cancer Center, 7701 Burholme Av, Philadelphia, PA 19111, USA.

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