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Blood, Vol. 113, Issue 3, 688-695, January 15, 2009
Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness
Blood Truksa et al.
113: 688
Supplemental materials for: Truksa et al
Files in this Data Supplement:
- Figure S1. A scheme of the deletions and mutations introduced into the human 2.3 to 2.5 Kb HAMP promoter (JPG, 202 KB)
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Murine Hamp1, Hamp2, and human HAMP were aligned and conserved transcriptional motifs were identified and deleted (A, B). Human promoter deletions are in blue while murine are in black. Similar to the murine Hamp1 mutations, human HAMP proximal STAT and distal HNF4α motif mutations were introduced (C) and tested for their responsiveness. The mutated nucleotides are underlined while the binding motifs are in bold.
- Figure S2. Mapping of SMAD1 (A), HJV (B), BMP-9 (C) responsive region of the human HAMP promoter (JPG, 46.2 KB)
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Firefly reporter constructs driven by the human HAMP promoter were used, 3.0 Kb HAMP WT was the positive full-length promoter control and pGL3 represented the negative promoterless control. The −2.3 to −2.5 Kb HAMP region is homologous to the −1.6 to −1.8 Kb region of murine Hamp1gene. Constructs were transfected into HepG2 cells and either co-transfected with SMAD1 (A) and hemojuvelin (B) or treated with 10ng/ml BMP9 (C) for 12–16 hrs. Firefly/renilla ratio of non-treated 3.0 Kb HAMP WT construct was set as 1.00. Means and SEM of the relative firefly/renilla ratios of at least three independent experiments are shown.
- Figure S3. The effect of BMP-RE1, BMP-RE2, and BMP-RE1/2 deletions on responsiveness to SMAD1, HJV, BMPs, and IL-6 (JPG, 78.4 KB)
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Constructs containing mutations in critical proximal 140–260bp, BMP-RE1, and distal BMP-RE2 and also the double mutant BMP-RE1/2 were introduced into the 2.5 Kb Hamp1 promoter reporter construct as described in Figure 3. Murine 2.5 Kb Hamp1 WT was the positive full-length promoter control and pGL3 represented the negative promoterless control. Control and mutant constructs were transfected into HepG2 cells and either co-transfected with SMAD1 or hemojuvelin or treated with 10ng/ml of IL-6, BMP-2, BMP4, or BMP9 for 12–16 hrs. Means and SEM of the relative firefly/renilla ratios and fold induction over control are shown.The total firefly/renilla ratio of untreated 2.5Kb Hamp1 WT construct was set as 1.00 and is depicted in panel A. The fold induction over control (non-treated cells), showing the ability to respond, is depicted in panel B.
- Figure S4. Effect of various HAMP promoter deletions on SMAD1 (A), HJV (B), BMP-9 (C) induced response (JPG, 46.6 KB)
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Firefly reporter constructs driven by the human HAMP promoter were used, 3.0 Kb Hamp1 WT was the positive full length promoter control and pGL3 represented the negative promoterless control. The proximal STAT mutation and A–D 50 bp deletions of HAMP distal −2.3 to −2.5 Kb region homologous to the −1.6 to −.8 Kb region of murine Hamp1 gene are described in Figure S1. Constructs were transfected into HepG2 cells and either co-transfected with SMAD1 (A) and hemojuvelin (B) or treated with 10ng/ml BMP9 (C) for 12–16 hrs. Firefly/renilla ratio of non-treated 3.0 Kb HAMP WT construct was set as 1.00. Means and SEM of the relative firefly/renilla ratios of at least three independent experiments are shown.
- Figure S5. Effect of distal BMP-RE and HNF4 motifs on SMAD1 (A), hemojuvelin (B) and BMP9 (C) induced response of the human HAMP promoter (JPG, 53.4 KB)
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Firefly reporter constructs driven by HAMP promoter were used, 3.0 Kb human HAMP WT was the positive full length promoter control and pGL3 represented the negative promoterless control. Constructs containing mutations in the distal HNF4A and HNF4B motifs were described in Fig. S1 and were transfected into HepG2 cells and either co-transfected with SMAD1 (A) and HJV (B) or treated with 10ng/ml BMP9 (C) for 12–16 hrs. Firefly/renilla ratio of non-treated 3.0 Kb human HAMP WT construct was set as 1.00. Means and SEM of the relative firefly/renilla ratios of at least three independent experiments are shown.
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