Divalent Metal Transporter 1 (DMT1) Regulates EPO Receptor Gene Expression Via GATA-1

Erythroid differentiation and iron metabolism are interconnected processes in order to produce sufficient numbers of appropriately hemoglobinized red cells. Patients carrying mutations of divalent metal transporter DMT1 display severe microcytic anemia and iron overload. In vivo, this defect can be partially rescued by stimulation of erythropoiesis by erythropoetin (EPO). In vitro, addition of EPO together with iron-saturated salicylaldehyde isonicotinoyl hydrazone (Fe-SIH, a non-transferrin iron donor) to the cultures significantly improved the growth of patient's DMT1-mutant erythroid progenitors (Horvathova et al, 2012). Regulation of erythropoiesis by EPO and its receptor (EPOR) involves transcription factor GATA-1. Our earlier data (Burda et al, 2009) showed that in erythroblasts GATA-1 transcriptionally regulates both Epor and Dmt1 and that this activation is blocked by negative regulator of erythroid differentiation, transcription factor PU.1. This suggests that simultaneous expression of EPOR and DMT1 is required for erythroid differentiation and survival of erythroid cells. We hypothesize that deficiency of DMT1 negatively affects expression of EPOR, thus leading to inhibition of EPO/EPOR signaling. We suppose that this inhibition involves GATA-1 and PU.1.

We used mouse erythroleukemia (MEL) cells expressing 17-OH-estradiol-inducible transgenes of GATA-1 (GER) or PU.1 (PUER). mRNA was quantitated by RT-PCR and protein occupancy on DNA was determined by chromatin immunoprecipitation (ChIP). Downregulation of Dmt1 was achieved by siRNA.

Using ChIP we established that GATA-1 and PU.1 regulate Epor gene directly. By activating GER or PUER in MEL cells we observed that promoter region of Epor is enriched and depleted respectively by acetylated histone H3 lysine (K) 9. Furthermore, inhibition of Epor by PU.1 coincided with recruitment of PU.1 to the Epor promoter. Similar ChIP analyses of Dmt1 promoter are undergoing. We next activated GER in MEL cells in the presence or absence of Dmt1 siRNA and observed that Dmt1 expression is needed for GATA-1-dependent upregulation of Epor mRNA expression during erythroid differentiation.

We found that GATA-1 stimulates both Epor and Dmt1 expression and that PU.1 blocks directly these effects. We also found that erythroid transcriptional regulation of Epor via transcription factor GATA-1 is severely diminished in Dmt1-knockdown MEL cells. We are currently testing whether this effect involves abnormal transport of iron or other divalent metals.

(Grant support: P305/11/1745, P301/12/P380, P305/12/1033. Institutional funding: PRVOUK-P24/LF1/3, UNCE 204021, SVV-2012–264507, GAUK 251135 82210).

No relevant conflicts of interest to declare.