Korean J Physiol Pharmacol.  2024 Mar;28(2):113-120. 10.4196/kjpp.2024.28.2.113.

Transcriptional regulation of genetic variants in the SLC40A1 promoter

• Ha SY
• Kim JY
• Choi JH

• KMID: 2553523
• DOI: http://doi.org/10.4196/kjpp.2024.28.2.113

Abstract

Solute carrier 40A1 (SLC40A1) encodes ferroportin, which is the only known transmembrane protein that exports elemental iron from mammalian cells and is essential for iron homeostasis. Mutations in SLC40A1 are associated with ironoverload disorders. In addition to ferroportin diseases, SLC40A1 expression is downregulated in various cancer types. Despite the clinical significance of the SLC40A1 transporter, only a few studies have investigated genetic variants in SLC40A1. The present study was performed to identify genetic variations in the SLC40A1 promoter and functionally characterize each variant using in vitro assays. We investigated four haplotypes and five variants in the SLC40A1 promoter. We observed that haplotype 3 (H3) had significantly lower promoter activity than H1, whereas the activity of H4 was significantly higher than that of H1. Luciferase activity of H2 was comparable to that of H1. In addition, four variants of SLC40A1, c.-1355G>C, c.-662C>T, c.-98G>C, and c.-8C>G, showed significantly increased luciferase activity compared to the wild type (WT), whereas c.-750G>A showed significantly decreased luciferase activity compared to the WT. Three transcription factors, cAMP response element-binding protein-1 (CREB-1), chicken ovalbumin upstream promoter transcription factor 1, and hepatic leukemia factor (HLF), were predicted to bind to the promoter regions of SLC40A1 near c.-662C>T, c.-98G>C, and c.-8C>G, respectively. Among these, CREB-1 and HLF bound more strongly to the variant sequences than to the WT and functioned as activators of SLC40A1 transcription. Collectively, our findings indicate that the two SLC40A1 promoter haplotypes affect SLC40A1 transcription, which is regulated by CREB-1 and HLF.

Keyword

Genetic variation; Promoter; SLC40A1; Transcription

Figure

• 

  Fig. 1 The effect of SLC40A1 promoter haplotypes or variants on its promoter activity. Luciferase activities were measured 48 h after transfection of reporter plasmids containing SLC40A1 haplotypes (A), or its variants (B) into HCT-116 cells. Relative luciferase activity of each haplotype or variant was compared to that of H1 or WT, respectively. Data shown represent mean ± SD from triplicate wells in a representative experiment. SLC40A1, solute carrier 40A1; WT, wild type. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 2 DNA-protein binding assays to examine the interaction between SLC40A1 promoter variants and transcription factors. Nuclear extracts from HCT-116 cells interacted with biotinylated oligonucleotide of CREB-1 consensus, c.-662C WT, or c.-662T variant (A) and biotinylated oligonucleotide COUP-TF1 consensus, c.-98G WT, or c.-98C variant (B) and biotinylated oligonucleotide of HLF consensus, c.-8C WT, or c.-8G variant (C). DNA-protein binding assay was performed using antibodies against CREB-1, COUP-TF1, or HLF. Data shown represent mean ± SD from three independent experiments. SLC40A1, solute carrier 40A1; WT, wild type; CREB-1, cAMP response element-binding protein-1; COUP-TF1, chicken ovalbumin upstream promoter transcription factor 1; HLF, hepatic leukemia factor; OD, optical density. *p < 0.05, **p < 0.01 vs. WT.

• 

  Fig. 3 The effect of transcription factors on the promoter activity of SLC40A1. Luciferase activities were measured 48 h after co-transfection of SLC40A1 WT or its variants and various amounts of CREB-1 (A), COUP-TF1 (B), or HLF (C) cDNA into HCT-116 cells. The luciferase activity of each construct was compared with naïve promoter activity. Data shown represent mean ± SD from triplicate wells in a representative experiment. SLC40A1, solute carrier 40A1; WT, wild type; CREB-1, cAMP response element-binding protein-1; COUP-TF1, chicken ovalbumin upstream promoter transcription factor 1; HLF, hepatic leukemia factor. **p < 0.01, ***p < 0.001 vs. naïve promoter activity.

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