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 IB isoforms of the divalent metal transporter (DMT 1) have recently been shown to be regulated transcriptionally via NF-kappa B 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 DMT 1. Promoter analysis has established that the I B 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 P 19 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 similar to 60% decrease in luciferase activity and similar to 65% decrease in I B 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 IB isoforms of DMT I mRNA.