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

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.