Expression of the 1B isoforms of divalent metal transporter (DMT1) is regulated by interaction of NF-Y with a CCAAT-box element near the transcription start site
✍ Scribed by Prasad N. Paradkar; Jerome A. Roth
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 169 KB
- Volume
- 211
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Abstract
The 1B isoforms of the divalent metal transporter (DMT1) have recently been shown to be regulated transcriptionally via NF‐κB but whether other regulatory elements are present on this promoter, however, have not been determined. Accordingly, studies were performed to delineate a minimal promoter region responsible for basal expression of these isoforms of DMT1. Promoter analysis has established that the 1B promoter is a TATA‐less promoter containing a common CCAAT‐box element conserved in mouse, rat, and human. Using luciferase reporter assays, it was found that mutation of this sequence leads to more than 95% reduction in the basal activity in mouse P19 cells. Using EMSA and ChIP assay, it was confirmed that NF‐YA protein subunit can bind specifically to this site. Transfecting these cells with a dominant negative (DN) form of NF‐YA leads to ∼60% decrease in luciferase activity and ∼65% decrease in 1B form of mRNA. To determine the location of the CCAAT‐box in relation to the transcription start site, 5′ RACE was performed. Results of these studies reveal that the CCAAT‐box resides at position −6 to −2 upstream from the transcriptional start site. These data demonstrate that binding of NF‐Y to this CCAAT‐box domain is responsible for the basal regulation of 1B isoforms of DMT1 mRNA. J. Cell. Physiol. 211: 183–188, 2007. © 2007 Wiley‐Liss, Inc.