Received for publication December 27, 2006.
Revised June 1, 2007.
Accepted for publication June 4, 2007.

Transcription Factor Binding to a Putative Double E-Box Motif Represses CYP3A4 Expression in Human Lung Cells

Abstract

Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5' upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, while the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57 base pair insertion in the CYP3A4 promoter region (-71 to -127), which was absent in the CYP3A5 promoter. Deletion of the 57 bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the ³²P-labeled CYP3A4-57 bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double or single E-box binding factors (TAL1, EF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5'-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5'-upstream region of the CYP3A5 gene, which attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case where a cytochrome P450 gene is actively repressed in a tissue-specific manner.

Key words: DNA binding sites, Promoter analysis, Regulation of gene expression, Cytochrome P450, Regulation - transcriptional