Mutation Of The Divalent Metal Transporter (Dmt1) Gene Results In Inefficient Induction Of The Erythroid Transcriptional Program Due To Latter Onset Of GATA-1 and Epor Expression
Authors: Curik Nikola , Burda Pavel , Zikmund Tomas , Savvulidi Filipp , Horvathova Monika , Strouboulis John , Divoky Vladimir , Stopka Tomas 
Published: October 2019

Abstract

Introduction
Divalent metal transporter 1 (DMT1; SLC11A2) encodes trans-membrane protein variants that execute either non-heme iron absorption through apical duodenal membrane of enterocytes or transferrin-bound iron uptake through endosomal membrane of erythroid (and other) cells. Since 2005 several DMT1 mutations affecting accurate protein folding and localization were identified in patients with defective iron uptake leading to microcytic anemia, abnormal growth of erythroid progenitors in vitro, and iron overload in the liver. We have previously demonstrated that defective growth of DMT-1-mutatnt BFU-Es in vitro and anemia associated with ineffective erythropoiesis in vivo can be improved with high-dose erythropoietin (EPO) supplementation (Horvathova et al., 2012). Data from Dmt1-mutant mk/mk mice (Gunshin et al., 2005) suggested that the anemic phenotype is a result of ineffective erythropoiesis within bone marrow and spleen.

Hypothesis
DMT1-mutant erythropoiesis inhibits EPO receptor (EPOR) signaling whose end-point target as well as upstream activator is the key transcription factor GATA-1. This results in defective erythroid development characterized by impaired survival capacity of erythroid progenitors, increased apoptosis of erythroblasts, and dysregulation of erythroid gene expression leading to ineffective erythropoiesis.

Materials
Bone marrow cells of murine mk/mk mice were sorted using flow cytometry to obtain differentiating erythroid fractions based on antigens Cd71 and Ter119 (Sokolovsky et al., 2001) which were then subjected to gene expression analysis.

Results
Firstly, four consecutive fractions (proerythroblasts and early basophilic erythroblasts - stage I, late basophilic erythroblasts – stage II, chromathophillic and orthochromathophillic erythroblasts – stage III, and late chromathophillic erythroblasts and reticulocytes – stage IV) were isolated from mk/mk mice and control littermates. Flow cytometry showed enrichment in stages I and II and depletion in stage IV in the mk/mk bone marrow when compared to wild type controls, consistent with our previous data. In the spleen the major cellular enrichment was seen in stage III accompanied by cell depletion in stage IV. Gene expression of GATA-1 was markedly decreased at the onset (in stages I and II) of erythropoiesis while it was increased in terminal stage IV. The expression pattern of the GATA-1 target gene Epor was similar to that of GATA-1 expression, while expression of b-maj globin was significantly reduced indicating developmental delay of the erythroid Dmt1-mutant compartment (compared to wild type). Whereas GATA-1 and Epor expression is low in early erythropoiesis, a compensatory increase in their expression at later stages is not capable to efficiently upregulate b-maj globin. These data together with flow cytometry analysis identify a developmental blockade of erythropoiesis between stages II(III) and IV. To better understand whether Dmt1 levels regulate GATA-1 and Epor expression we used murine erythroleukemia (MEL) cells containing conditional (estrogen-regulated) transgene encoding GATA-1 fused with estrogen receptor ligand-binding domain (GER). MEL cells are cytologically characterized as proerythroblasts and early basophilic erythroblasts, stage I. Firstly, we established that GATA-1 upregulates Epor expression and directly binds to the Epor gene using RT-PCR, Immunoblotting, and chromatin immunoprecipitation (ChIP) in activated GER cells. Using ChIP-sequencing analysis of GATA-1 (and a panel of histone modifications) the GATA-1 enrichment was clearly identified at three distinct Epor regions in murine erythroblasts and differentiating MEL cells. Next, we downregulated Dmt1 using siRNA and observed that GATA-1-mediated upregulation of Epor in activated GER cells became inhibited. In addition, the knockdown of Dmt1 also inhibited steady state levels of GATA-1 in MEL cells by 25%.

Conclusions
Ineffective erythropoiesis in Dmt1-mutant mice is blocked at stages II (III) and display deregulation of the Epor signaling cascade involving GATA-1 and its targets. Our data thus interconnect iron uptake and the Epor/GATA-1 pathways and suggest their roles during erythroid pathogenesis upon DMT1 mutations. Grants: P305/11/1745, P301/12/P380, P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, GAUK 251135 82210

Disclosures:
No relevant conflicts of interest to declare.