Association of SMAD3/4, COUP-Tf, HNF4α, C/EBPα, GATA2 and STAT5 to the Hamp1 Promoter

There are two regions of the murine Hamp1 promoter that have been shown to be critical for Hamp1 expression. The 260 bp proximal region and the distal −1.6 to −1.8 Kb regions appear to be required for responsiveness to IL-6, BMPs and iron. Analyses of 160 bp proximal promoter for consensus transcription factor motifs by MatInspector identified a STAT5 site at the location identified previously by Wrighting et al., Blood 2006, as a functional STAT3 site and by Courselaud et al., J Biol Chem 2002, as a C/EBPα site. Although a SMAD responsive site was not predicted in this region, we (in press), and Verga-Falzacappa et al., J Mol Med 2008, have demonstrated that there is a functional BMP responsive element (GGCGCC) in this region. A probe encompassing the putative BMP-RE1, STAT, C/EBPα, and AP1 motifs were used in electrophoretic mobility shift assays (EMSA). We found that the addition of cold competitor DNA corresponding to STAT3, C/EBPα and AP1 consensus motifs did not block the binding of transcription factors from liver nuclear extracts to the BMP-RE1/STAT/C/EBPα/AP1 probe. In contrast, the addition of cold competitor DNA corresponding the SMAD3/4 or STAT5 completely blocked essentially all binding of liver nuclear transcription factors to the BMP-RE1/STAT/C/EBPα/AP1 probe. Analyses of the −161 to −260 bp proximal promoter for consensus transcription factor motifs identified a GATA2 binding site and a SMAD responsive site (TGTCTGCCC). Two long probes encompassing the to −161 to −260 bp region were used in EMSAs. Binding of liver nuclear extracts to a probe encompassing the GATA motif was blocked by the addition of a GATA consensus DNA. Similarly, binding to a long probe encompassing the SMAD responsive site was blocked by the addition of a SMAD3/4 consensus DNA. Analyses of the 1.6 to 1.7 Kb region of the distal murine Hamp1 promoter identified several transcription factor motifs: bZIP transcription factor that acts on nuclear genes encoding mitochondrial proteins, COUP-Tf/HNF4α, and MEL1 (MDS1/EVI1-like gene1) to be both in human and mouse Hamp genes. Although a SMAD responsive site was not identified in this region, we have demonstrated that there is a functional BMP responsive element (GGCGCC) in this region. Using EMSA with probes corresponding to the −1.6 to −1.7 bp region of the hepcidin promoter, we examined the binding of transcription factors from liver nuclear extracts derived from mice. Binding of liver nuclear extract to a probe corresponding to the BMP-RE2, bZIP, HNF4α, COUP motifs was blocked by cold competitor probes corresponding to SMAD3/4, HNF4α, COUP-Tf, and Stat5. Whereas competitor probes to Smad3/4 and HNF4α competed for the binding of specific bands to the radiolabelled probe, total binding was blocked with cold competitor probes to the consensus COUP-Tf and Stat5 motifs. Supershift analyses using antibodies to HNF4α, COUP, SMAD4 demonstrated the binding of these transcription factors to the radiolabeled BMP-RE2/bZIP/HNF4α/COUP probe. Binding to a probe encompassing a MEL motif was blocked by the addition of cold competitor to C/EBPα and could be supershifted with antibodies against C/EBPα. In conclusion, SMAD 3/4, COUP-Tf, HNF4α, C/EBPα, GATA2 and STAT5 appear to be important in the regulation of Hamp1 expression.