Regulation of erythroid maturation by the transcriptional repressor NKAP

 描述(申请人提供)：红细胞在生物体的整个生命周期中不断产生。虽然多能祖细胞进入红系的受体和转录调节因子(如GATA-1)已被很好地描述，但调节红系成熟后期的分子却鲜为人知。只有在红细胞生成素调节的早期红系细胞增殖爆发后，细胞周期停止后，成熟才开始。在红细胞阶段，血红素化需要铁代谢。在红细胞成熟之前，染色质浓缩也是随后的红细胞去核所必需的。我们发现，我们实验室首次从功能上定义的转录抑制因子NKAP对于红细胞生成是绝对必要的。在红细胞生成素受体(“ErGFP-cre NKAP CKO小鼠”)的控制下，使用CRE的红系血统中NKAP的特异性缺失会导致胚胎在大约11-12天的胚胎死亡。胚胎11.5天时，ErGFP-cre NKAP CKO胚胎贫血，面色苍白。流式细胞仪分析表明，在CD71+Ter119红系阶段，红细胞的生成不会超过CD71+Ter119。因此，NKAP对于红细胞生成是绝对必要的。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ABCB7的表达减少。ABCB7将铁-S-谷胱甘肽簇从线粒体转运到胞浆中，在胞浆中，铁-S簇被结合到蛋白质中，如血红蛋白。在没有ABCB7的情况下，铁-S簇聚集在线粒体中，普鲁士蓝染色可以检测到这一点。与ABCB7表达降低一致，有条件的NKAP缺失会导致细胞内铁的异常积聚。为了确定在缺乏NKAP的情况下，ABCB7的减少是否可能导致红细胞生成的阻断，我们产生了ABCB7的红系特异性缺失(ErGFP-cre ABCB7 CKO)。与ErGFP-cre NKAP CKO胚胎类似，ErGFP-cre ABCB7 CKO胚胎大约在胚胎E11-12死亡，面色苍白，贫血，在CD71+Ter119红细胞阶段以外的红细胞生成受阻。因此，ErGFP-cre NKAP CKO胚胎和ErGFP-cre ABCB7 CKO胚胎的红细胞生成阻断之间的表型差异表明ABCB7可能是NKAP调控红细胞生成的关键基因。这项建议的重点是了解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.