Transcriptional regulation of chicken cytochrome P450 2D49 basal expression by CCAAT/enhancer-binding protein α and hepatocyte nuclear factor 4α

Chicken cytochromeP450 (CYP)2D49 is structurally and functionally related to human CYP2D6, which is an important drug-metabolizing enzyme. To date, little is known about the transcriptional regulation of this cytochrome. Through deletion analysis of the CYP2D49 promoter, we identified two putative degenerate CCAAT/enhancer-binding protein (C/EBP)-binding sites and an imperfect DR1 element (the site contains direct repeats of the hexamer AGGTCA separated by a one-nucleotide spacer motif) within regions -296/-274, -274/-226, and -226/-183, respectively, which may play critical roles in the transcriptional activation of the CYP2D49 gene. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the putative C/EBP boxes and DR1 element in the CYP2D49 promoter are functional motifs that bind to C/EBP and hepatocyte nuclear factor4 (HNF4), respectively. Furthermore, we studied the functional importance and relationships of these transcription factor-binding sites by examining the effects of mutation and deletion of these regions on promoter activity. These studies revealed that the two C/EBP-binding sites show a compensatory relationship and work cooperatively with the DR1 element to modulate the transcription of CYP2D49. The results of overexpressing C/EBP and HNF4 in culture cells further confirmed that both C/EBP and HNF4 contribute significantly to sustaining a high level of CYP2D49 transcription. In conclusion, the data indicate that the constitutive hepatic expression of CYP2D49 is governed by both C/EBP and HNF4. Further studies will be required to fully characterize the molecular mechanisms that modulate CYP2D49 expression.