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