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

Chicken cytochrome P450 (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/EBPα and hepatocyte nuclear factor 4α (HNF4α), 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/EBPα and HNF4α in culture cells further confirmed that both C/EBPα and HNF4α 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/EBPα and HNF4α. Further studies will be required to fully characterize the molecular mechanisms that modulate CYP2D49 expression.