pkm2 Binds to the Regulatory Regions of Gata-1 and stat5 in β-Thalassemic Mouse Erythroblasts

β-thalassemia is a worldwide distributed hereditary red cell disorder characterized by chronic anemia due to the combination of reduced red cell survival and ineffective erythropoiesis. Clinical management of β-thalassemic (β-thal) patients is primarily based on red blood cell transfusion and iron-chelation therapy. We previously showed increased expression of pyruvate kinase M2 (PKM2) in both mouse and human β-thal erythroblasts when compared to healthy ones, as a possible adaptative mechanism against chronic oxidation. Studies in other disease models have shown that PKM2 might acquire nuclear localization as phospho-PKM2 (p-PKM2, Ser 37), associating with different transcriptional factors such as GATA. Here, we analyzed sorted erythroid precursors (CD44⁺TER119⁺Fsc^High cells) from bone marrow of wild-type (n=8) and Hbb^th3/+ (β-thal, n=6) mice. In β-thal erythroid precursors, we found increased p-PKM2 when compared to wild-type erythroblasts. To better understand p-PKM2 distribution, we used immunomicroscopy with specific anti-PKR and anti-p-PKM2 antibodies. In both wild-type and β-thal erythroid precursors, PKR was similarly localized in the cytoplasm of erythroblasts. Conversely, we found increased amount of p-PKM2 as large clusters in the nucleus of β-thal erythroid precursors when compared to wild-type cells. To understand whether PKM2 binds to regulatory regions of erythropoiesis relevant genes, we performed Chromatin Immunoprecipitation (ChIP) assay using specific anti-PKM2 antibody in sorted erythroid cells from both mouse strains. In β-thal erythroblasts, PKM2 binds to site in the promoter regions of Gata1 and Stat5. We noted that in β-thal erythroid precursors, the binding of PKM2 on Gata1 promoter results in the upregulation of Gata1 expression, the association of PKM2 with that of Stat5 leads to Stat5 downregulation when compared to wild-type mice. In addition, PKM2- binding sites on Stat5 promoter appear to colocalize with NF-kB consensus motifs, suggesting a possible functional synergy between these two transcription factors. This is extremely interesting since STAT5 is involved in cell responsiveness to erythropoietin and GATA1 contributes to cell differentiation during erythropoiesis. Furthermore, the crosstalk between STAT5 and NF-kB suggests the possible involvement of pro-oxidant and pro-inflammatory pathways in β-thal ineffective erythropoiesis. In conclusion, in β-thal mouse erythroblasts we show for the first time nuclear compartmentalization of PKM2 when compared to healthy cells. Our data demonstrate that PKM2 binds to regulatory regions of Gata1 and Stat5. This later appears to synergize with NF-kB in β-thal mouse erythroblast, suggesting a novel crosstalk between STAT5 and NF-kB in ineffective β-thal erythropoiesis.