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 5Ј 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, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (Ϫ71 to Ϫ127) that 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 32 P-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 5Ј-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5Ј-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.

Zinc ions upregulate the hormone gastrin via an E-box motif in the proximal gastrin promoter

“…Our result that Zn 2C ions induce gastrin expression through the activation of an E-box binding transcription factor via the phosphorylation of AKT raises the possibility that Zn 2C may induce the EMT through direct activation of E-boxbinding transcription factors. Although further studies of the transcription factors involved are warranted, in the light of evidence that many known (Longo et al 2008, Terragni et al 2011 and as yet uncharacterized transcription factors (Biggs et al 2007) can bind E-boxes it is beyond the scope of the current study to identify one single transcription factor which activates gastrin expression in response to Zn 2C . What does our finding that gastrin is induced by Zn 2C ions imply about normal gastric physiology?…”

Xenobiotic-Induced Transcriptional Regulation of Xenobiotic Metabolizing Enzymes of the Cytochrome P450 Superfamily in Human Extrahepatic Tissues

“…On the other hand, CYP3A4 was found in bronchial glands, bronchiolar columnar and terminal epithelium, type II alveolar epithelium, and alveolar macrophages, although only about 20% of individuals was positive in the immunostaining [153]. Recently, a putative double E-box repressor motif in the 5'-upstream region of the CYP3A4, but not CYP3A5 promoter, has been discovered, which attenuates CYP3A4 expression in the human lung cell line A549 cells derived from type II alveolar pneumocyte [169]. Induction of CYP3A5 and CYP3A4 genes was also examined in A549 adenocarcinoma cell line.…”

Pregnane X Receptor and Yin Yang 1 Contribute to the Differential Tissue Expression and Induction of CYP3A5 and CYP3A4

“…The binding sites for previously characterized transcriptional regulators CCAAT-box, ER6, BTE, TATA-box, and NF1 [37], [38], [39] are underlined. The portion of the NF1 binding site described to constitute a CCAAT box, the YY1 site, and the two E-box motifs [27], [39], all contained in the 57 bp region, are boxed. The positions of binding sites are shown separately for CYP3A4 and, if applicable, in brackets for CYP3A5 .…”

“…As no such excessive activity was observed with the “spacer” sequence ( CYP3A5 -Spins, Figure 3B), this suggested the existence within the 57 bp fragment of additional, as yet unidentified transcriptional enhancers which come to light after the removal of the YY1-mediated repression. However, mutant CYP3A5 -57M6, generated to inactivate a putative E-box-like binding site [27] overlapping with the YY1-binding site (Figure 2), had no effect on activity. Likewise, we saw no changes in luciferase activity upon the mutation of the NF1 or of the second, more downstream E-box binding site (Figure S1).…”

“…The non-expression of CYP3A4 as opposed to CYP3A5 in a lung-derived cell line has been linked to a 57 bp insertion into the gene's promoter, but the exact mechanism has not been identified [27]. The expression of CYP3A5 in the prostate has been reported to be mediated by a promoter element binding the androgen receptor [10].…”