Abstract
Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.
Highlights
Hypoxia-inducible factor 2␣ (HIF2␣) directly regulates a battery of genes essential for intestinal iron absorption
Our data demonstrate that SMAD family member 3 (SMAD3) and SMAD4 are iron-regulated transcription factors that are decreased following iron deficiency, leading to a derepression and optimization of HIF2␣-dependent iron absorption
We show that SMAD3 and SMAD4 are iron-regulated transcription factors that limit HIF2␣ transcriptional activity under normal cellular iron
Summary
HIF2␣ modulators were assessed using a high-throughput siRNA screen for genes that regulate the Dmt promoter Both SMAD3 and SMAD4 significantly suppressed HIF2␣-induced DMT1 promoter activity that contained 0.2 kb of the proximal promoter This suggests the possibility of novel interaction(s) with transcription factors that bind in this 0.2-kb region (Fig. 5D). The suppression of DMT1 activity by SMAD4 was completely abolished by S4R81A and S4K88A mutants (Fig. 6C) These data indicate that the SMAD4 DNA binding is essential for the suppression of HIF2␣ iron target genes. Dmt and Dcytb were robustly potentiated following 1 week of low-iron diet (Fig. 7B) These data were consistent with protein-level changes, DMT1 was significantly elevated in the Smad4⌬IE mice, and DCYTB, not significant, demonstrated an increase in expression compared with littermate controls (Fig. 7C)
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