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-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.
Paradkar
Prasad N
PN
Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14214, USA.
Roth
Jerome A
JA
D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D024363:Transcription Initiation Site; D000099236:Solute Carrier Family 11, Member 2
D023081:CCAAT-Binding Factor; D027682:Cation Transport Proteins; D017931:DNA Primers; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger; D000099236:Solute Carrier Family 11, Member 2