Figure 4.
Loss of Tet1 reduces FPN degradation and Rnf217 promoter demethylation. (A) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated for 20 hours either with or without FAC (100 µM), then treated for 2 hours with CHX (75 µM) followed by hepcidin (0.5 µM, 5 hours); FPN was then measured using western blot analysis (n = 4 mice per group). (B) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated with FAC (100 µM, 20 hours), then treated for 4 hours with CHX (75 µM), CHX + MG132 (10 µM), or CHX + CQ (100 µM), after which FPN was measured using western blot analysis (n = 4 mice per group). (C) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated for 20 hours with FAC (100 µM), followed by MG132 (10 µM, 4.5 hours); the cells were then treated with hepcidin (1 mg/mL) for 0, 20, or 40 minutes. The cell lysates were immunoprecipitated using an anti-FPN antibody and immunoblotted using the FK2 anti-polyubiquitin antibody. The red asterisk labels the polyubiquitinated FPN (C). (D) Quantification of FPN ubiquitination (n = 4 per group). (E) Schematic diagram depicting the main steps in screening for potential E3 ligase genes. (F) Interaction between FPN and RNF217 measured using the Y2H screening, indicated by survival of the harboring colonies in SD-4 medium (lacking Ura, His, Leu, and Trp) and SD-2 medium (lacking Ura and His). AD, activation domain; BD, binding domain. (G) Co-IP assay to identify the putative interaction between Flag-tagged RNF217 and Myc-tagged FPN expressed in HEK293T cells. Red asterisks indicate the expected size of Myc-tagged FPN (top) and Flag-tagged RNF217 (bottom). (H) Relative mRNA levels of the indicated E3 ligase genes were measured in BMDMs (n = 4 per group) and the duodenum (n = 5-8 mice per group) of Tet1+/+ and Tet1−/− mice under iron-overload conditions. (I) Relative Rnf217 mRNA levels were measured in Tet1+/+ and Tet1−/− BMDMs treated for the indicated times with 100 µM FAC (n = 3 per group). (J) Relative levels of 5-hmC at 3 regions in the Rnf217 promoter in Tet1+/+ and Tet1−/− BMDMs treated with FAC (100 µM) (n = 4 per group). (K) Methylation in the Rnf217 promoter was measured using bisulfite Sanger sequencing in FAC-treated Tet1+/+ and Tet1−/− BMDMs (n = 4 per group). Closed circles indicate methylated sites. *P < .05 and **P < .01 (Student t test).

Loss of Tet1 reduces FPN degradation and Rnf217 promoter demethylation. (A) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated for 20 hours either with or without FAC (100 µM), then treated for 2 hours with CHX (75 µM) followed by hepcidin (0.5 µM, 5 hours); FPN was then measured using western blot analysis (n = 4 mice per group). (B) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated with FAC (100 µM, 20 hours), then treated for 4 hours with CHX (75 µM), CHX + MG132 (10 µM), or CHX + CQ (100 µM), after which FPN was measured using western blot analysis (n = 4 mice per group). (C) BMDMs from Tet1+/+ and Tet1−/− mice were pretreated for 20 hours with FAC (100 µM), followed by MG132 (10 µM, 4.5 hours); the cells were then treated with hepcidin (1 mg/mL) for 0, 20, or 40 minutes. The cell lysates were immunoprecipitated using an anti-FPN antibody and immunoblotted using the FK2 anti-polyubiquitin antibody. The red asterisk labels the polyubiquitinated FPN (C). (D) Quantification of FPN ubiquitination (n = 4 per group). (E) Schematic diagram depicting the main steps in screening for potential E3 ligase genes. (F) Interaction between FPN and RNF217 measured using the Y2H screening, indicated by survival of the harboring colonies in SD-4 medium (lacking Ura, His, Leu, and Trp) and SD-2 medium (lacking Ura and His). AD, activation domain; BD, binding domain. (G) Co-IP assay to identify the putative interaction between Flag-tagged RNF217 and Myc-tagged FPN expressed in HEK293T cells. Red asterisks indicate the expected size of Myc-tagged FPN (top) and Flag-tagged RNF217 (bottom). (H) Relative mRNA levels of the indicated E3 ligase genes were measured in BMDMs (n = 4 per group) and the duodenum (n = 5-8 mice per group) of Tet1+/+ and Tet1−/− mice under iron-overload conditions. (I) Relative Rnf217 mRNA levels were measured in Tet1+/+ and Tet1−/− BMDMs treated for the indicated times with 100 µM FAC (n = 3 per group). (J) Relative levels of 5-hmC at 3 regions in the Rnf217 promoter in Tet1+/+ and Tet1−/− BMDMs treated with FAC (100 µM) (n = 4 per group). (K) Methylation in the Rnf217 promoter was measured using bisulfite Sanger sequencing in FAC-treated Tet1+/+ and Tet1−/− BMDMs (n = 4 per group). Closed circles indicate methylated sites. *P < .05 and **P < .01 (Student t test).

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