TfR1 and Ferrochelatase Expression During Erythroid Differentiation Induced by 5-Aza-Cdr In Both MEL Cells and BFU-E-Derived Erythroblasts Correlates with c-Myc Function

Abstract 4248

During erythroid differentiation, heme biosynthesis increases dramatically and this is accompanied by a marked increase in transferrin receptor 1 (TfR1) expression and iron uptake from transferrin. It has long been known that 5-aza-2′deoxycytidine (5-aza-CdR), a demethylating agent, is able to induce murine erythroid leukemia (MEL) cell differentiation. However, the detailed mechanism underlying this induction remains elusive. In the current study, we report that 5-aza-CdR induces erythroid differentiation in MEL cells and BFU-E-derived erythroblasts and that this is associated with the nuclear translocation of the transcription factor c-Myc and its binding to Max to form functional complex. As markers of erythroid differentiation, the expression levels of TfR1, ferrochelatase and ALAS2 were analyzed by quantitative real-time PCR and western blotting. TfR1 and ferrochelatase expression were increased 5-fold following 5-aza-CdR treatment. To determine whether these changes in gene expression reflected differential methylation, we analyzed 1.5 kb up- and downstream from the transcription start site (TS) of these genes. Although many potential methylation sites (CpG islands) were found in both the TfR1 and ferrochelatase genes, bisulfite sequencing revealed that few sites (< 5%) were methylated in these promoters in untreated MEL cells and that there was no difference following 5-aza-CdR treatment. This observation suggests that the increased expression of TfR1 and ferrochelatase is not directly mediated by 5-aza-CdR-induced DNA demethylation. EKLF and GATA1 are key transcription regulators in erythroid differentiation, and the iron regulatory proteins (IRPs) are important for the post-transcriptional regulation of TfR1 mRNA levels. Bioinformatic analysis showed that there were many potential methylation sites in the EKLF and IRP2 promoters, however, bisulfite sequencing showed that there was no effect of 5-aza-CdR treatment at these sites in MEL cells. Since c-Myc has been reported to stimulate TfR1 expression, we then investigated the role of c-Myc in erythroid differentiation. Levels of c-Myc mRNA and protein were not increased by 5-aza-CdR, however, we did observe a dramatic increase in the nuclear fraction of c-Myc. Furthermore, co-immunopricipitation studies showed that the amount of the Myc-Max complex, which is the form in which c-Myc activates downstream gene expression, increased in the nucleus after 5-aza-CdR treatment. Interestingly, using chromatin-immunoprecipitation we also observed very strong binding of c-Myc to the CACGTG sequence upstream of the TfR1 transcription start site following 5-aza-CdR treatment. Analogous studies using primary mouse erythroblasts derived from BFU-Es gave similar results, with 5-aza-CdR treatment leading to increased TfR1 and ferrochelatase expression, c-Myc translocation to the nucleus and increased c-Myc binding activity to TfR1 promoter. Taken together, these studies point to an important role for c-Myc in the regulation of TfR1 during erythroid differentiation induced by demethylation. Precisely how demethylation is linked to alterations in c-Myc activity is under investigating.