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

Abstract

The 1B isoforms of the divalent metal transporter (DMT1) have recently been shown to be regulated transcriptionally via NF-kappaB 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 approximately 60% decrease in luciferase activity and approximately 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.

Increased Hippocampal Expression of the Divalent Metal Transporter 1 (DMT1) mRNA Variants 1B and +IRE and DMT1 Protein After NMDA-Receptor Stimulation or Spatial Memory Training

“…Knowledge of differential transcriptional regulation of DMT1 expression is emerging. Both the inflammatory cytokine nuclear factor kappa B (NFjB) and the nuclear factor Y (NF-Y) regulate DMT1-1B expression in embryonic carcinoma cells (Paradkar and Roth 2007). In contrast, hypoxia upregulates expression of the 1A mRNA variant in PC12 cells, presumably through activation of hypoxic response elements in its promoter region (Lis et al 2005).…”

Regulatory mechanisms of intestinal iron absorption—Uncovering of a fast‐response mechanism based on DMT1 and ferroportin endocytosis

“…Treatment of Caco‐2 cells with the iron chelator desferioxamine for 36 h resulted in moderate increase in DMT1 and FPN1 gene transcription, whereas incubation with FeCl 3 led to decreased DMT1 and FPN transcription [28]. The proinflammatory agent LPS and the cytokines TNFα, IFN‐γ, and transcription factor NF‐κB induce DMT1 expression, indicating a link between inflammation and DMT1 expression [32, 33]. The 1B isoform of DMT1 has a NFκB‐responsive element in its 5′ untranslated region, whereas the 1A isoform contains Hif‐2α response elements that regulate intestinal DMT1 in response to hypoxia [34, 35].…”

Parkin regulates metal transport via proteasomal degradation of the 1B isoforms of divalent metal transporter 1

“…Transcriptionally regulated (Hubert and Hentze 2002) splice variants, exon 1A and 1B, have also been identified on the 5¢-end of the message . Exon 1B is spliced out of the message containing exon 1A but is incorporated into the untranslated region of the message in the 1B isoforms of DMT1 accounting for the fact that translation of the 1B mRNAs actually begins at exon 2 (Paradkar and Roth 2007). Although the selective physiological functions of the four isoforms of DMT1 have not been defined, all are capable of transporting iron and other transition metals and all have similar kinetic properties (Garrick et al 2006a,b;Mackenzie et al 2007).…”