Association of SMAD3/4, COUP-Tf, HNF4α, C/EBPα, GATA2 and STAT5 to the Hamp1 Promoter
Authors: Lee Pauline , Truksa Jaroslav , Beutler Ernest 
Published: October 2019

Abstract
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.