Impact of TET2 Deficiency on Iron Metabolism in Erythroblasts: A Potential Link to Ring Sideroblast Formation
Authors: Inokura Kyoko , Fujiwara Tohru , Okitsu Yoko , Fukuhara Noriko , Onishi Yasushi , Ishizawa Kenichi , Shimoda Kazuya , Harigae Hideo 
Published: October 2019

Abstract
Background: Sideroblastic anemia is characterized by the presence of ring sideroblasts (RS) in the bone marrow (BM). RS are caused by iron accumulation in the mitochondria of erythroblasts and are seen in both acquired and congenital forms of sideroblastic anemia. Although dysfunction in mitochondrial metabolism has been implicated in the pathogenesis of sideroblastic anemia, the true mechanism leading to RS formation remains elusive. Clonal sideroblastic anemia is usually acquired in the context of myelodysplastic syndrome (MDS): The presence of 15% or more RS in the BM with appropriate morphologic and cytogenetic criteria for MDS is best classified as refractory anemia with ring sideroblasts (RARS), but varying quantities of RS (<15%) can also occur in refractory anemia with multilineage dysplasia (RCMD) and other myeloid malignancies as well. Intriguingly, a subset of MDS as well as myeloid malignancies has been reported to harbor a somatic mutation of the TET2gene (Delhommeau F et al., 2009; Malcovati et al., 2014), which regulates DNA demethylation by hydroxylating 5-methylcytosine to 5-hydroxymethylcytosine. Thus, because TET2 has also been shown to play an important role during erythroid differentiation (Pronier et al., 2011), TET2 may be involved in iron metabolism and/or heme biosynthesis in erythroblasts, contributing to the formation of RS. To explore this possibility, we conducted biological and molecular analyses based on TET2-knockdown mice.
Methods: TET2-knockdown mice (Ayu17-449, Tet2trap) were generated by gene trap project (Shide et al., 2012). Four-month-old heterozygous Tet2trap/+ mice were used in the present study. Serum levels of iron, total iron-binding capacity, unbound-iron binding capacity and ferritin were measured by enzyme-linked immunosorbent assay. BM cells were stained for CD71 and Ter119 (BD Pharmingen, San Jose, CA, USA), and sorted into I–IV populations according to a previously reported method (Socolovsky et al., 2001). To obtain Ter119+ erythroblasts, a magnetic cell sorting system was used (Miltenyi Biotec, Auburn, CA, USA). Western blot analysis was performed with anti-mitochondrial ferritin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Prussian blue staining was used to assess iron deposition in erythroblasts. DNA methylation status was assessed with the EpiXploreTM Methylated DNA Enrichment Kit (Clontech, Mountain View, CA, USA). Statistical significance was assessed by the two-sided ttest, and p value of <0.05 were considered statistically significant.
Results: Tet2trap/+ mice at 4 months exhibited mild normocytic anemia in comparison to the wild-type (WT) mice (hemoglobin; 14.5 ± 1.1 and 13.2 ± 0.6 g/dl, for WT and Tet2trap/+, respectively, p < 0.05, n = 8). Concomitantly, the number of cells in erythroid populations III–IV (polychromatic to orthochromatic erythroblasts) was decreased. Interestingly, serum iron and ferritin levels were significantly elevated in Tet2trap/+ mice (ferritin; 104.7 ± 42.4 and 171.6 ± 89.5 ng/ml, for WT and Tet2trap/+, respectively, p < 0.05, n = 8). Western blot analysis confirmed that the amount of mitochondrial ferritin was increased in Ter119+ erythroblasts from Tet2trap/+ mice compared with those from WT mice. On the other hand, BM from Tet2trap/+ mice did not show an increased number of RS. To explore the molecular mechanism by which TET2 deficiency induces iron overload, quantitative reverse transcription–polymerase chain reaction analysis was conducted with Ter119+ cells for genes involved in erythroid differentiaition, heme biosynthesis and iron metabolism. The analysis demonstrated significant downregulation of heme oxygenase 1 (Hmox1), ferrochelatase (Fech) and Tet2 in Tet2trap/+ mice, whereas the expression of erythroid-specific 5-aminolevulinate synthase (Alas2), mitoferrin (Slc25a37) and Gata1 were unchanged. Because a CpG island was idenitified in the promoter of Fech (http://genome.ucsc.edu), we evaluated its DNA methylation status and found that the CpG site of Fech shows significantly high methylation in Ter119+ cells of Tet2trap/+ mice compared with those of WT mice. As FECH catalyzes the insertion of ferrous iron into the protoporphyrin IX to produce heme, the reduced expression of Fech by TET2 deficiency may result in inhibiton of heme synthesis, leading to iron overload in mitochondria.
Conclusion: TET2 is involved in iron and heme metabolism in erythroblasts.

Disclosures
Fujiwara: Chugai Pharmaceutical CO., LTD.: Research Funding.