Regulation of erythroid maturation by the transcriptional repressor NKAP

 描述（由申请方提供）：红细胞在生物体的整个生命周期中持续产生。虽然多能祖细胞进入红系谱系的受体和转录调节因子（如加塔-1）已被充分表征，但调节红系成熟后期的分子却鲜为人知。成熟仅在细胞周期停滞后开始，随后在早期红系细胞中促红细胞生成素调节的增殖爆发。在成红细胞阶段，血红蛋白化需要铁代谢。在成为成熟红细胞之前，成红细胞的后续去核也需要染色质浓缩。我们已经发现，在我们的实验室，NKAP，转录抑制因子的功能首先定义，是绝对需要红细胞生成。在促红细胞生成素受体的控制下使用cre在红细胞系中特异性缺失NKAP（“ErGFP-cre NKAP cKO小鼠”）导致大约在胚胎第11-12天的胚胎致死。在胚胎第11.5天，ErGFP-cre NKAP cKO胚胎贫血且苍白.流式细胞仪分析表明，红细胞生成不进行超过CD 71 + Ter 119 hi成红细胞阶段。因此，NKAP是红细胞生成所必需的。我们的初步数据表明，在ST-HSC/多能祖细胞中NKAP的缺失导致线粒体内膜蛋白ABCB 7的表达降低。ABCB 7将Fe-S-谷胱甘肽簇从线粒体转运到细胞质中，在那里Fe-S簇被并入蛋白质如血红蛋白中。在不存在ABCB 7的情况下，Fe-S簇在线粒体中积累，这可以使用普鲁士蓝染色检测。与ABCB 7表达降低一致，NKAP的条件性缺失导致细胞内铁的异常积累。为了确定在不存在NKAP的情况下ABCB 7的减少是否可能导致红细胞生成的阻断，我们产生了ABCB 7的红细胞特异性缺失（ErGFP-cre ABCB 7 cKO）。与ErGFP-cre NKAP cKO胚胎相似，ErGFP-cre ABCB 7 cKO胚胎在大约胚胎e11-12死亡，苍白且贫血，并且在CD 71 + Ter 119 hi成红细胞阶段之后具有红细胞生成阻滞。因此，在ErGFP-cre NKAP cKO胚胎和ErGFP-cre ABCB 7 cKO胚胎中的红细胞生成阻滞之间的表型相似性表明ABCB 7可能是NKAP在红细胞生成中调控的关键基因。该提案的重点是了解NKAP如何调节红细胞生成。

 DESCRIPTION (provided by applicant): 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.