Transcriptional Regulation of Hepcidin by Iron.

Abstract

Transcriptional regulation by iron in mammalian systems is poorly understood. Hepcidin, a 25 amino acid peptide that plays a central role in iron homeostasis, is transcriptionally regulated by iron. A region of the murine hepcidin promoter 1.6 to 1.8 kb upstream from the start of translation was recently identified to be important in transcriptional regulation by iron (Truksa J, et al. The distal location of the iron responsive region of the hepcidin promoter. Blood DOI 10.1182/blood-2007-05-091108, 2007). In order to identify transcription factors that might be important in regulation by iron, transcription factor microarray analyses (Panomics TranSignal Protein/DNA Array) were performed with nuclear extracts from livers of mice made iron deficient or iron loaded for 4 weeks. The analyses revealed 43 transcription factors that were upregulated in iron loaded liver nuclear extracts and 39 transcription factors that were upregulated in iron deficient nuclear extracts. In the region of the promoter we had found essential for transcriptional regulation by iron, −1.6 to −1.8 kb, consensus motifs were identified by Genomatix MatInspector for 10 transcription factors that corresponded to transcription factors upregulated in high iron nuclear extracts by array analyses. Similarly, the consensus sequences for 5 transcription factors corresponded to transcription factors identified in iron deficient nuclear extracts. Electrophoretic mobility shift assays were performed with probes across this region of the murine hepcidin promoter. Several probes exhibited differential binding between deficient and high iron nuclear extracts. These include the probe encompassing the CCAAT box and MEL1 motif, a probe containing a HLH motif, and a probe containing a bZIP and COUP motif. The probe containing the CCAAT motif was supershifted with antibodies against CBF, but was not supershifted with antibodies against SMAD4, CEBPα, and COUP. The probe containing a bZIP and COUP motif can be supershifted with antibodies against COUP-Tf and HNF4α, but not with antibodies against SMAD4, CEBPα, and COUP. Our data suggest that CBFA, COUP, and HNF4α are involved in transcriptional regulation of hepcidin by iron.