【摘要】：造血生成是由少量的造血干細胞(HPCs)通過增殖分化產(chǎn)生多種成熟血細胞的過程,造血系統(tǒng)作為研究分化相關(guān)細胞分子機制的模型被廣泛關(guān)注。正常造血分化是個復(fù)雜多步驟的過程,受到細胞間和細胞內(nèi)多種信號的嚴密調(diào)控,這些信號最終靶向轉(zhuǎn)錄調(diào)控因子,特異轉(zhuǎn)錄因子的表達決定了細胞的分化方向。造血轉(zhuǎn)錄因子的缺失或功能異常會導(dǎo)致造血分化出現(xiàn)問題,引發(fā)造血系統(tǒng)疾病。因此對造血轉(zhuǎn)錄因子的表達及功能的精確調(diào)控在正常造血生成過程中具有重要意義。 紅細胞是造血生成中產(chǎn)生最多的細胞類型,紅系細胞生成的轉(zhuǎn)錄調(diào)控是造血系統(tǒng)中研究較多的一類。紅系分化是一個受到精密調(diào)節(jié)的有序過程,與動態(tài)的特異基因表達模式密切相關(guān),目前已發(fā)現(xiàn)許多轉(zhuǎn)錄因子和信號通路參與其中。在紅系造血分化過程中,紅系特異基因漸次開啟表達,而另一些基因的表達逐漸關(guān)閉,最終細胞呈現(xiàn)紅系特異表型。 GATA-1是紅系分化和成熟過程中的中心轉(zhuǎn)錄因子,GATA-1的靶基因有很多,幾乎所有的紅系特異表達的基因都是GATA-1的靶基因,GATA-1既可以作為激活子也可以作為抑制子起作用。GATA-1還能與多種轉(zhuǎn)錄因子及輔因子相互作用,其特定功能與其相互作用因子密切相關(guān)。發(fā)現(xiàn)新的GATA-1的相互作用蛋白并對其進行研究有利于我們更好地理解GATA-1的功能以及紅系分化過程中的轉(zhuǎn)錄調(diào)控機制。 早幼粒細胞白血病蛋白PML是腫瘤抑制子,主要定位于PML核體內(nèi),PML蛋白的核體內(nèi)定位,對于其功能的發(fā)揮和核體的形成至關(guān)重要。PML核體可通過募集和釋放多種蛋白參與多種細胞功能的調(diào)控,例如誘導(dǎo)細胞衰老和凋亡,增殖抑制,維持基因組穩(wěn)定性和抗病毒。此外,PML在造血系統(tǒng)中亦具有重要作用,PML參與造血祖細胞分化,還有研究表明PML參與粒系分化,而PML在紅系分化中的作用還未被充分闡釋。 基因的表達模式往往與其功能相關(guān),我們首先用realtime-PCR的方法檢測了PML在發(fā)育不同階段紅細胞中的表達情況,結(jié)果發(fā)現(xiàn)PML在8.5d-10.5d的卵黃囊中表達較低,而在11.5d-14.5d的胎肝中表達較高,說明PML在小鼠紅系發(fā)育較晚期組織中高表達,且表達譜與GATA-1存在一致性。同時,以G1E-ER4, MEL, K562細胞系作為誘導(dǎo)紅系分化的模型,檢測了PML在分化不同階段紅系細胞中的表達,PML在誘導(dǎo)紅系分化過程中持續(xù)表達,在EPO誘導(dǎo)CD34+細胞向紅系分化時表達上調(diào),且同樣發(fā)現(xiàn)PML與GATA-1的表達譜存在一致性。隨后我們構(gòu)建了PML和GATA-1的表達質(zhì)粒,利用co-IP, GST pull down等方法證明體內(nèi)和體外PML能與GATA-1直接相互作用,免疫熒光結(jié)果說明PML可以將GATA-1募集到PML核體上。接著我們構(gòu)建了PML和GATA-1的系列缺失突變體,同樣用co-IP的方法證明分別是GATA-1的C端鋅指和PML的coiled-coil結(jié)構(gòu)域介導(dǎo)了它們的相互作用。 PML與GATA-1的相互作用是否是功能性的呢?通過報告基因?qū)嶒灠l(fā)現(xiàn),PML可以增強GATA-1的轉(zhuǎn)錄活性,且PML與GATA-1的相互作用對于PML增強GATA-1的轉(zhuǎn)錄活性必不可少。PML是如何影響GATA-1的轉(zhuǎn)錄活性的呢?我們利用報告基因、IP、ChIP方法對其機制進行研究發(fā)現(xiàn),PML增強體內(nèi)GATA-1與DNA的結(jié)合,PML促進GATA-1與其輔因子p300的協(xié)同激活能力,并可促進GATA-1與p300的相互作用,增強p300對GATA-1的乙�；�,增加CBP/p300在GATA-1結(jié)合位點上的募集。 我們用PML的誘導(dǎo)劑干擾素α處理K562細胞,發(fā)現(xiàn)PML表達上調(diào)的同時,珠蛋白基因的表達也上調(diào)。進一步在K562細胞中過表達PML可檢測到細胞增殖抑制,細胞周期G1期阻滯,紅系分化標志物珠蛋白和血型糖蛋白A等的表達上調(diào),干擾PML則這些基因表達下調(diào),而GATA-1過表達對珠蛋白的影響與PML類似。同樣,PML過表達的G1E-ER4細胞中,α,β-珠蛋白轉(zhuǎn)錄增強,聯(lián)苯胺染色結(jié)果顯示血紅蛋白生成增多,說明PML促進紅系分化成熟。我們還在人原代紅細胞中進行了PML的過表達和干擾,發(fā)現(xiàn)原代細胞中PML也可以增強珠蛋白基因的表達。此外,利用GATA-1缺失的G1E細胞和GATA-1活性可誘導(dǎo)的G1E-ER4細胞為研究模型,我們發(fā)現(xiàn)PML對紅系分化成熟的促進作用是GATA-1依賴的。 綜上所述,我們的研究首次發(fā)現(xiàn)PML可作為GATA-1新的調(diào)節(jié)因子,將GATA-1募集到PML核體,促進GATA-1與CBP/p300的協(xié)同作用,增強GATA-1的乙�；腕w內(nèi)與DNA的結(jié)合,從而提高GATA-1的轉(zhuǎn)錄活性,并且通過調(diào)節(jié)GATA-1的活性參與紅系終末分化。
[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
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

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