【作者】 丁亚丽；

【导师】 杨晓明； 李长燕；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 红系分化相关基因EDAG (Erythroid Differentiation-Associated Gene)是一种特异表达于造血组织和细胞,对造血系统的发育及分化起重要调控作用的基因。目前对EDAG的作用机制尚缺乏深入的了解。本论文从基因调控、功能结构域、相互作用蛋白质三个方面对EDAG发挥作用的分子机制进行初步探讨。第一,EDAG调控造血细胞分化的分子机制研究。在小鼠髓系前体细胞32D细胞中过表达EDAG导致红系和巨核系表型的出现。利用小鼠全基因组芯片分析基因表达变化,发现过表达EDAG可导致32D细胞的332个基因表达下调,288个基因表达上调。其中红系／巨核系转录因子GATA-1及其靶基因EKLF、hemoglobin、NF-E2、Gfi-1b等基因的表达明显上调,而肥大细胞表面标记GP49A和单核细胞特异标记CD14等则表现为下调表达。在K562细胞中采用RNAi敲低EDAG水平,GATA-1及其靶基因NF-E2、Gfi-1b、HBB、EKLF和EPX的mRNA表达水平均显著下调。这些结果表明EDAG的高表达可促进32D细胞向红系／巨核系分化,转录因子GATA-1可能在此过程中起重要作用。第二,EDAG蛋白质功能与结构的研究。通过双荧光素酶报告基因检测,发现Gal4DBD-EDAG融合蛋白质在哺乳动物细胞中表现出转录抑制活性,但EDAG的124-184aa和384-484aa这两段区域具有较强的转录激活活性。转录抑制因子CtBP1可抑制124-184aa和384-484aa的转录激活活性。EDAG在PMA刺激的条件下,第123位和381位的丝氨酸磷酸化水平上调,但这两个磷酸化位点的突变并不影响EDAG的转录调节活性。提示EDAG同时具有转录激活和转录抑制活性,其调控基因表达的机制可能非常复杂。第三,EDAG与NPM相互作用的初步研究。通过Co-IP验证EDAG与NPM存在生理性相互作用,EDAG通过其N末端(1-124aa)与NPM的N末端(1-187aa)相互作用。在PMA诱导的K562巨核分化过程中,过表达EDAG可稳定NPM蛋白质,而下调表达EDAG则加速NPM蛋白质的下调。EDAG对NPM稳定性的影响可能参与EDAG对造血细胞分化、增殖、存活的调控过程。更多还原

【Abstract】 Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we investigate the function and molecular mechanism of EDAG through three aspects.First, we studied the molecular mechanism of EDAG in hematopoietic cell differentiation by identifying genes regulated by overexpressing EDAG in a myeloid cell line 32D. Overexpression of EDAG in 32D cells led to an erythroid/ megakaryocytic phenotype. Using a genome-wide microarray analysis and a two-fold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, hemoglobin, NF-E2, Gfi-lb etc. were increased. While monocyte differentiation antigen CD 14 and mast cell surface glycoprotein GP49A were decreased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of GATA-1 and its target genes. These results suggested that EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.Second, we focused on indentifying the function and structure of EDAG. Using the dual-luciferase reporter assay, Gal4DBD-EDAG fusion protein was found as a transcriptional repressor in different mammalian cells. However, two transactivation domains (124-184aa and 384-484aa) was characterized and showed significant transcriptional activation activity. Co-expression of transcriptional repressor CtBP1 repressed the transactivaiton activity of two transactivation domains. Furthermore, two serine sites (S123 and S381) can be phosphorylated under treatment with PMA, but mutation of these amino acids had no effect on the activity of EDAG. These results indicated that EDAG both has transactivation and repression activity, and the mechanism by which it regulates gene expression is more complicated. Third, we characterized NPM as a physiological binding partner of EDAG. EDAG was shown to interact with the N-terminal (1-187aa) of NPM through its N-terminal (1-124aa) region, and stabilized NPM protein. During PMA-induced K562 megakaryocytic differentiation, overexpression of EDAG prevented the down-regulation of NPM proteins, while knockdown of EDAG enhanced the degradation of NPM.更多还原