去泛素化酶USP7调控GATA1影响红系终末分化机制研究

The precise regulation of key proteins in erythroid differentiation is essential for the maintenance of normal differentiation. The deubiquitination enzymes play an important roles in the regulation of protein levels. The preliminary data show that the ubiquitination enzyme USP7 increases during human terminal erythroid differentiation, the USP7 has been reported to regulate a variety of important biological processes, but its function in the erythropoiesis is unknown. Our preliminary data showed knock down of USP7 resulted in terminal erythroid differentiation delay, proliferation inhibition and apoptosis induction. Furthermore, we found that USP7 can positively regulate the key transcription factor GATA1 protein level and USP7 interacts with GATA1. In addition, we also found that compared with health donor, USP7 was decreased in β-thalassemia (β-TM) patients. Based on the preliminary results we propose the hypothesis that USP7 is essential for maintaining normal erythropoiesis through stabilizing GATA1; in β-TM, down-regulation of USP7 decreases GATA1 protein level, resulting in erythroid differentiation block and increased apoptosis, which is a possible cause of ineffective erythropoiesis. In this study, we will confirm the scientific hypothesis through some experiments carried out in normal case and β-TM patient samples and some mice, which will help clarify the functions of USP7 in erythroid differentiation and enrich the regulation mechanisms of GATA1 and provide new targets for ineffective erythropoiesis in β-TM.

红系分化中关键蛋白的精准调控是正常分化的前提，蛋白水平调控中去泛素化酶有重要功能，本课题前期数据显示去泛素化酶USP7在人红系分化终末阶段显著增加，已报道USP7可调控多种重要生命活动，但在红系分化中功能未知。我们发现敲低USP7延迟终末红系分化、抑制增殖及诱导凋亡；进一步发现USP7正调控红系核心转录因子GATA1蛋白表达水平并与其存在相互作用，且USP7在β-地中海贫血（β-TM）病人中表达下调。基于以上结果，我们提出如下科学假说：USP7是红系发育正常进行的必需分子，USP7去泛素化稳定GATA1维持红系发育正常进行；在β-TM患者中USP7下调引起GATA1下降导致红系分化阻滞和凋亡增加是无效造血的原因之一。本项目将在正常人与β-TM病人样本及小鼠中验证该科学假说，明确USP7在红系分化中的功能，丰富GATA1的调控机制，为治疗β-TM无效造血提供新靶标。

红系分化过程中细胞组成成分会发生剧烈变化，大量蛋白降解, 同时也伴随着蛋白的稳定, 它们之间的动态平衡需要精确调控以维持正常分化。在红系发育过程中蛋白降解主要是由泛素-蛋白酶体通路介导，泛素化是一个可逆过程，去泛素化酶（Deubiquitinases, DUBs）能够特异性将泛素分子从蛋白上水解下来，逆转泛素化过程, 从而稳定细胞内蛋白水平。但目前在红系分化中蛋白稳定的调控机制研究较少。本课题利用本团队前期获得的红系分化各阶段细胞转录组数据，发现泛素特异性蛋白酶家族(Ubiquitin-specific proteases，USPs)成员USP7在红系分化过程中显著上调，提示USP7在红系分化中具有重要功能。USP7在多种生命过程中发挥重要功能，但其在红系发育中的功能尚未见报道。本课题旨在探讨去泛素化酶USP7在红系发育中的作用和机制。重要结果和数据：1.USP7在红系发育中具有重要功能。通过使用USP7的shRNA和特异性抑制剂在体外诱导红系分化体系中将USP7敲低或抑制，发现终末红系分化显著延迟，血红蛋白表达下降。同时USP7抑制剂能延迟小鼠贫血恢复，表明USP7在红系分化中功能重要。2. USP7通过调控GATA1影响红系发育。利用USP7 shRNA及特异性抑制剂在红系分化体系中敲低或抑制USP7，发现红系发育关键转录因子GATA1蛋白水平显著下调。进一步发现USP7与GATA1共定位于核中且存在直接相互作用并直接调控GATA1的K48链连接类型泛素化影响GATA1蛋白的稳定性。3. USP7通过稳定乙酰化的GATA1影响GATA1功能。GATA1乙酰化促进GATA1与DNA结合，进一步的磷酸化可导致GATA1发生泛素化降解，终止GATA1的转录。USP7与GATA1乙酰化相关，GATA1的C末端乙酰化可影响USP7与GATA1的结合。科学意义：本研究首次揭示了去泛素化酶参与终末红系发育调控，去泛素化酶USP7能调控终末红系发育，鉴定了红系分化中关键转录因子GATA1是USP7的底物，阐明USP7通过稳定乙酰化的GATA1影响GATA1功能。丰富了GATA1的调控机制，为红系分化的调控提供了新思路，有助于进一步理解GATA1紊乱相关疾病的发病机制及开发新的治疗策略。