LSD1介导的表观遗传修饰调控GATA Switch的机制研究

发布时间：2018-04-20 22:00

本文选题：LSD1 + 组蛋白去甲基化　；参考：《吉林大学》2016年博士论文

【摘要】：背景:LSD1是第一个被发现的组蛋白赖氨酸特异性去甲基化酶,它的发现使人们重新定义了组蛋白去甲基化这一可逆的过程,它以一种FAD依赖性的氧化反应方式特异性的催化H3K4位点的单甲基化及二甲基化的去甲基化,且可以和TAL1等多种转录因子以复合体形式对靶基因的表达发挥调控功能,在造血系统分化过程中发挥重要作用。包括GATA1、GATA2多种转录因子所形成的调控网络对于维持正常的造血细胞分化起到了至关重要的作用,GATA2主要表达于造血干/祖细胞,并随造血细胞分化表达下调,而GATA1表达含量上调,进而调控造血细胞的增殖和分化(GATA Switch),GATA1、GATA2这种表达含量的动态变化趋势对于造血系统分化具有重要意义,目前对于GATA Switch机制的研究主要集中于GATA1、GATA2互相的调控功能。目的:从表观遗传角度出发,探究组蛋白甲基化在造血分化过程中的影响,从表观遗传修饰层面上对调控造血细胞分化的机制作进一步阐释。GATA1、GATA2以自身蛋白表达量动态变化呈现的GATA Switch更是具有深远的研究意义。因LSD1介导的表观遗传修饰作用可以调节多种造血相关靶基因的转录水平,研究LSD1对GATA Switch的调控机制成了探讨表观遗传修饰作用调控造血系统分化的重要着力点。选取人的红系白血病细胞K562、小鼠的红系白血病细胞MEL为细胞模型,通过诱导细胞向红细胞方向分化,动态完整地分析在造血细胞分化不同时期LSD1所介导的表观遗传修饰作用对GATA Switch的影响,从组蛋白修饰这一全新的角度阐释GATA Switch的调控机制,为我们进一步探索造血系统分化过程中的调控机制提供理论依据及新的研究方向,为阐明疾病中表观遗传修饰变化以及基因转录调控的分子机制奠定基础。方法:通过免疫共沉淀IP实验鉴定了和GATA2发生相互作用的LSD1复合体,比较了在造血细胞分化的不同时期,LSD1、TAL1、GATA2之间的相互作用水平的变化情况;构建、表达并纯化了具有不同结构域的GST-LSD1N-term、GST-LSD1N-term+SWIRM、GST-LSD1SWIRM、GST-LSD1AO、GST-LSD1SWIRM+AO融合蛋白,通过GST-pull down Assay,具化了LSD1和GATA2之间相互作用的结构域;通过RT-PCR检测了缺失LSD1对GATA转录因子表达水平的影响;通过染色质免疫共沉淀Ch IP等技术揭示了LSD1复合体在造血分化的不同时期作用于不同的GATA基因位点,分析了造血分化过程中GATA1、GATA2基因位点组蛋白甲基化水平的变化,探讨了LSD1所介导的组蛋白去甲基化功能对于靶基因表达水平、造血细胞分化带来的影响。结果:通过GST-pull down Assay、免疫共沉淀IP、染色质免疫共沉淀Ch IP等技术揭示了GATA2可以和LSD1复合体相互作用,LSD1通过SWIRM结构域与GATA2相互作用。造血分化早期GATA2募集LSD1至GATA1基因db G、IE位点,进而抑制GATA1表达;随造血细胞分化,GATA2和LSD1之间相互结合变弱,TAL1和LSD1之间结合水平增强,TAL1将LSD1招募至GATA2基因的1S启动子位点,进而抑制GATA2表达,促进红细胞分化。LSD1通过调节GATA1、GATA2蛋白表达情况对造血系统分化发挥重要调控功能,LSD1、TAL1、GATA2以复合体形式动态调控这一过程。结论:从一个全新的组蛋白修饰的角度阐释了GATA Switch的调控机制,明确了LSD1所介导的表观遗传学修饰对GATA Switch的调控功能,从而建立了LSD1调控造血分化机制的新模型。
[Abstract]:Background: LSD1 is the first found histone lysine specific demethylation enzyme. Its discovery makes people redefine the reversible process of histone demethylation, which catalyzes the monomethylation of H3K4 loci and demethylation of the two methylation with a FAD dependent oxidation reaction, and can be more than TAL1 and so on. The transcription factor plays an important role in the expression of the target gene in the form of complex, which plays an important role in the differentiation of hematopoietic system. The regulatory network, including GATA1, GATA2, plays a vital role in maintaining normal hematopoietic cell differentiation, and GATA2 is mainly expressed in hematopoietic stem / progenitor cells. The expression of hematopoietic cells is down regulated and the expression of GATA1 is up-regulated, and then the proliferation and differentiation of hematopoietic cells (GATA Switch), and the dynamic trend of GATA1 and GATA2 expression content is important for the differentiation of hematopoietic system. At present, the research on GATA Switch mechanism mainly focuses on GATA1 and GATA2 mutual regulation function. From the epigenetic point of view, the effect of histone methylation on the process of hematopoietic differentiation is explored..GATA1 is further interpreted from the epigenetic modification level to regulate the differentiation of hematopoietic cells. The GATA Switch presented by GATA2 in the dynamic changes of its own protein expression is of profound significance. The apparent remains of LSD1 The effect of modification can regulate the transcriptional level of a variety of hematopoietic target genes. The study of the regulatory mechanism of LSD1 on GATA Switch has become an important point to explore the effect of epigenetic modification on the regulation of hematopoietic differentiation. The human erythroleukemia cell K562 is selected and the erythroleukemia cell MEL in mice is the cell model, and the cells are induced to be red. The cell direction differentiation, dynamic and complete analysis of the effect of epigenetic modification on GATA Switch mediated by LSD1 in different stages of hematopoietic differentiation, explains the regulatory mechanism of GATA Switch from the new angle of histone modification, and provides a theoretical basis for us to further explore the regulation mechanism in the process of hematopoietic system differentiation. The research direction is the basis for elucidating the changes in epigenetic modification and the molecular mechanism of gene transcription regulation in the disease. Methods: the LSD1 complex interacting with GATA2 was identified by the immunoprecipitation IP experiment. The changes in the level of interaction between LSD1, TAL1 and GATA2 at different stages of the differentiation of hematopoietic cells were compared. Construction, expression and purification of GST-LSD1N-term, GST-LSD1N-term+SWIRM, GST-LSD1SWIRM, GST-LSD1AO, GST-LSD1SWIRM+AO fusion protein with different domains, through GST-pull down Assay, with the interaction between LSD1 and GATA2; through RT-PCR detection of the effect of missing LSD1 on the expression level of transcription factors; The technique of chromatin immunoprecipitation Ch IP revealed that the LSD1 complex acted on different GATA loci in different periods of hematopoiesis differentiation, and analyzed the changes in the methylation level of the GATA1, GATA2 gene loci during the hematopoietic differentiation, and discussed the expression level of the target gene and the hematopoiesis mediated by the histone demethylation function mediated by LSD1. The effect of cell differentiation. Results: GST-pull down Assay, immunoprecipitation IP, chromatin immunoprecipitation Ch IP and other techniques reveal that GATA2 can interact with LSD1 complex, LSD1 interacts with GATA2 by SWIRM domain. With the differentiation of hematopoietic cells, the combination of GATA2 and LSD1 weakens and the binding level between TAL1 and LSD1 is enhanced. TAL1 recruits LSD1 to the 1S promoter site of the GATA2 gene, and then inhibits GATA2 expression, and promotes the proliferation of erythrocyte differentiation.LSD1 by regulating GATA1, and GATA2 protein expression plays an important role in the differentiation of the blood making system. This process is dynamically regulated by the complex form. Conclusion: the regulatory mechanism of GATA Switch is explained from a new histone modification point of view, and the regulatory function of epigenetic modification mediated by LSD1 is clarified, and a new model for the regulation of hematopoietic differentiation by LSD1 is established.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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