PML4通过增强GATA-1转录活性参与红系分化成熟

发布时间：2019-05-28 13:20

【摘要】：造血生成是由少量的造血干细胞(HPCs)通过增殖分化产生多种成熟血细胞的过程,造血系统作为研究分化相关细胞分子机制的模型被广泛关注。正常造血分化是个复杂多步骤的过程,受到细胞间和细胞内多种信号的严密调控,这些信号最终靶向转录调控因子,特异转录因子的表达决定了细胞的分化方向。造血转录因子的缺失或功能异常会导致造血分化出现问题,引发造血系统疾病。因此对造血转录因子的表达及功能的精确调控在正常造血生成过程中具有重要意义。红细胞是造血生成中产生最多的细胞类型,红系细胞生成的转录调控是造血系统中研究较多的一类。红系分化是一个受到精密调节的有序过程,与动态的特异基因表达模式密切相关,目前已发现许多转录因子和信号通路参与其中。在红系造血分化过程中,红系特异基因渐次开启表达,而另一些基因的表达逐渐关闭,最终细胞呈现红系特异表型。 GATA-1是红系分化和成熟过程中的中心转录因子,GATA-1的靶基因有很多,几乎所有的红系特异表达的基因都是GATA-1的靶基因,GATA-1既可以作为激活子也可以作为抑制子起作用。GATA-1还能与多种转录因子及辅因子相互作用,其特定功能与其相互作用因子密切相关。发现新的GATA-1的相互作用蛋白并对其进行研究有利于我们更好地理解GATA-1的功能以及红系分化过程中的转录调控机制。早幼粒细胞白血病蛋白PML是肿瘤抑制子,主要定位于PML核体内,PML蛋白的核体内定位,对于其功能的发挥和核体的形成至关重要。PML核体可通过募集和释放多种蛋白参与多种细胞功能的调控,例如诱导细胞衰老和凋亡,增殖抑制,维持基因组稳定性和抗病毒。此外,PML在造血系统中亦具有重要作用,PML参与造血祖细胞分化,还有研究表明PML参与粒系分化,而PML在红系分化中的作用还未被充分阐释。基因的表达模式往往与其功能相关,我们首先用realtime-PCR的方法检测了PML在发育不同阶段红细胞中的表达情况,结果发现PML在8.5d-10.5d的卵黄囊中表达较低,而在11.5d-14.5d的胎肝中表达较高,说明PML在小鼠红系发育较晚期组织中高表达,且表达谱与GATA-1存在一致性。同时,以G1E-ER4, MEL, K562细胞系作为诱导红系分化的模型,检测了PML在分化不同阶段红系细胞中的表达,PML在诱导红系分化过程中持续表达,在EPO诱导CD34+细胞向红系分化时表达上调,且同样发现PML与GATA-1的表达谱存在一致性。随后我们构建了PML和GATA-1的表达质粒,利用co-IP, GST pull down等方法证明体内和体外PML能与GATA-1直接相互作用,免疫荧光结果说明PML可以将GATA-1募集到PML核体上。接着我们构建了PML和GATA-1的系列缺失突变体,同样用co-IP的方法证明分别是GATA-1的C端锌指和PML的coiled-coil结构域介导了它们的相互作用。 PML与GATA-1的相互作用是否是功能性的呢?通过报告基因实验发现,PML可以增强GATA-1的转录活性,且PML与GATA-1的相互作用对于PML增强GATA-1的转录活性必不可少。PML是如何影响GATA-1的转录活性的呢?我们利用报告基因、IP、ChIP方法对其机制进行研究发现,PML增强体内GATA-1与DNA的结合,PML促进GATA-1与其辅因子p300的协同激活能力,并可促进GATA-1与p300的相互作用,增强p300对GATA-1的乙酰化,增加CBP/p300在GATA-1结合位点上的募集。我们用PML的诱导剂干扰素α处理K562细胞,发现PML表达上调的同时,珠蛋白基因的表达也上调。进一步在K562细胞中过表达PML可检测到细胞增殖抑制,细胞周期G1期阻滞,红系分化标志物珠蛋白和血型糖蛋白A等的表达上调,干扰PML则这些基因表达下调,而GATA-1过表达对珠蛋白的影响与PML类似。同样,PML过表达的G1E-ER4细胞中,α,β-珠蛋白转录增强,联苯胺染色结果显示血红蛋白生成增多,说明PML促进红系分化成熟。我们还在人原代红细胞中进行了PML的过表达和干扰,发现原代细胞中PML也可以增强珠蛋白基因的表达。此外,利用GATA-1缺失的G1E细胞和GATA-1活性可诱导的G1E-ER4细胞为研究模型,我们发现PML对红系分化成熟的促进作用是GATA-1依赖的。综上所述,我们的研究首次发现PML可作为GATA-1新的调节因子,将GATA-1募集到PML核体,促进GATA-1与CBP/p300的协同作用,增强GATA-1的乙酰化和体内与DNA的结合,从而提高GATA-1的转录活性,并且通过调节GATA-1的活性参与红系终末分化。
[Abstract]:Hematopoietic production is a process of producing a plurality of mature blood cells by the proliferation and differentiation of a small number of hematopoietic stem cells (HPCs), and the hematopoietic system is widely concerned as a model for the research and differentiation-related cellular molecular mechanism. The normal hematopoiesis differentiation is a complex multi-step process, which is controlled by various signals in the cell and in the cell, which finally target the transcription regulation factor, and the expression of the specific transcription factor determines the differentiation direction of the cells. The deficiency or abnormal function of the hematopoietic transcription factor can lead to the problems of hematopoietic differentiation and cause the system of hemopoietic system diseases. Therefore, the precise regulation of the expression and function of the hematopoietic transcription factor is of great significance in the process of normal hematopoiesis. The red blood cell is the most important cell type in the generation of hematopoietic cells, and the transcription regulation of the erythroid cell is more research in the hemopoietic system. The differentiation of the red system is a well-regulated and orderly process, which is closely related to the dynamic specific gene expression pattern, and many transcription factors and signal pathways have been found to be involved. in that proces of the differentiation of the red system, the specific gene of the red system is gradually open, and the expression of the other genes is gradually close, and the final cell is characterized in that the red system is specific Phenotype. GATA-1 is a central transcription factor in the process of erythroid differentiation and maturation. The target gene of GATA-1 is a lot. Almost all of the genes specifically expressed in the red system are the target genes of GATA-1. GATA-1 can be used as an activator or as a suppressor. GATA-1 can interact with various transcription factors and cofactors, its specific function and its interaction. It is closely related that the new GATA-1 interacting protein is found and its research is helpful for us to better understand the function of GATA-1 and the transformation in the process of erythroid differentiation. The promyelocytic leukemia protein PML is a tumor suppressor, which is mainly located in the PML nucleus, and is located in the nucleus of the PML protein, and plays an important role in the function of the PML. The formation of the body is critical. The PML nucleus may be involved in the regulation of various cell functions by raising and releasing a variety of proteins, such as inducing cell senescence and apoptosis, proliferation inhibition, maintenance of the gene, In addition, PML plays an important role in hematopoietic system, and PML is involved in the differentiation of hematopoietic progenitor cells. The expression pattern of the gene is often related to its function. We first detect the expression of PML in different stages of red blood cells by using the method of realtime-PCR, and the results show that the expression of PML in the yolk sac of 8.5 d-10.5d is low, while at the time of 11.5 d-14 The expression of PML in the fetal liver of the mice was high, and the expression profile of PML was high in the advanced tissues of the mouse red system. GATA-1 was consistent. At the same time, the expression of PML in the erythroid cells of different stages of differentiation was detected with G1E-ER4, MEL, and K562 cell lines. Up-regulation of the expression of the cells in the erythroid system, and also found that PML and GATA The expression profile of PML and GATA-1 was confirmed by co-IP and GST pull down. The results showed that PML could directly interact with GATA-1. -1 was raised onto the PML nucleus. Then we constructed a series of deletion mutants of PML and GATA-1, and the co-IP method was also used to demonstrate the coiled-coil structure of the C-terminal zinc finger and PML, respectively, of GATA-1. The domains mediate their interactions. PML and GATA- Whether the interaction of PML and GATA-1 can enhance the transcription activity of GATA-1, and the interaction of PML with GATA-1 has been found to be a function of PML-enhanced G. The transcriptional activity of ATA-1 is essential. PML is how to shadow In response to the transcriptional activity of GATA-1, the mechanism of GATA-1 is studied by using the reporter gene, IP, and ChIP method. The combination of GATA-1 and DNA in PML is enhanced, and the co-activation of GATA-1 with its cofactor p300 is promoted, and the interaction between GATA-1 and p300 can be promoted. 300 for GATA-1, increase CBP/ p300 The recruitment of GATA-1 binding site. We treated K562 cells with an inducer of PML, and found the expression of PML. At the same time, the expression of the globin gene is up-regulated. The expression of PML in K562 cells can be further detected, such as cell proliferation inhibition, cell cycle G1 arrest, erythroid differentiation marker globin and blood group glycoprotein A, and the expression of these genes is down-regulated, while GATA- The effect of over-expression on the globin is similar to that of PML. In the same way, in the G1E-ER4 cells that are overexpressed in the PML, the transcription of the antigen, the HCO3-globin is enhanced, and the staining result of the benzidine shows that the hemoglobin is born. in addition, that PML promote the differentiation and maturation of the red system. we also carried out the over-expression and the interference of PML in human primary red blood cells, and found that the primary cells In addition, the GATA-1-deleted G1E cells and GATA-1 activity-inducible G1E-ER4 cells were used as the research models. In conclusion, we have found that PML can be used as a new regulation factor for GATA-1, and GATA-1 is raised to PML nucleus to promote the synergistic effect of GATA-1 and CBP/ p300. A is combined to improve the transcription activity of GATA-1 and
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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