Erythrocytes are continuously produced throughout the life of an organism. While the receptors and transcriptional regulators for the entry of multipotent progenitors into the erythroid lineage (such as GATA-1) are well characterized, the molecules that regulate the later stages of erythroid maturation are less well known. Maturation initiates only after cell cycle arrest subsequent to an erythropoietin-regulated burst of proliferation in early erythroid cells. At the erythroblast stage, iron metabolism is required for hemoglobinization. Chromatin condensation is also required for subsequent enucleation of erythroblasts prior to becoming mature red blood cells. We have found that a transcriptional repressor first defined functionally in our laboratory, NKAP, is absolutely required for erythropoiesis. Specific deletion of NKAP in the erythroid lineage using cre under the control of the erythropoietin receptor (ErGFP-cre NKAP cKO mice) results in embryonic lethality at approximately embryonic day 11-12. At embryonic day 11.5, ErGFP- cre NKAP cKO embryos are anemic and pale. FACS analysis demonstrated that erythropoiesis does not proceed beyond the CD71+Ter119hi erythroblast stage. Therefore, NKAP is absolutely required for erythropoiesis. Our preliminary data indicated that deletion of NKAP in ST-HSC/multipotent progenitors led to decreased expression of the mitochondrial inner membrane protein ABCB7. ABCB7 transports Fe-S-glutathione clusters from the mitochondria into the cytosol, where Fe-S clusters are incorporated into proteins such as hemoglobin. In the absence of ABCB7, Fe-S clusters accumulate in the mitochondria, which can be detected using Prussian Blue staining. Consistent with decreased expression of ABCB7, conditional deletion of NKAP results in abnormal accumulation of intracellular iron. To determine whether decreased ABCB7 could be responsible for the block in erythropoiesis in the absence of NKAP, we generated an erythroid specific deletion of ABCB7 (ErGFP-cre ABCB7 cKO). Similar to ErGFP-cre NKAP cKO embryos, ErGFP-cre ABCB7 cKO embryos die at approximately embryonic e11-12, are pale and anemic, and have a block in erythropoiesis beyond the CD71+Ter119hi erythroblast stage. Thus, the phenocopy between the block in erythropoiesis in ErGFP-cre NKAP cKO embryos and ErGFP-cre ABCB7 cKO embryos indicate that ABCB7 may be the critical gene regulated by NKAP during erythropoiesis. The focus of this proposal is to understand how NKAP regulates erythropoiesis.
Continuous erythropoiesis is required throughout the lifetime of an organism for the generation of new red blood cells. These studies are directly relevant to human health, as defect in erythropoiesis are responsible for anemias. In particular, defects in iron homeostasis can lead to refractory anemias with ringed sideroblasts (RARS) for which therapeutic options are few, except for regular blood transfusions for the rest of the patient's lie. We have shown that the transcriptional repressor NKAP is critical for erythropoiesis. Understanding the role of NKAP in erythropoiesis may identify new targets for therapeutic intervention to treat anemia.
