组蛋白脱甲基酶LSD1在小鼠胚胎干细胞分化及斑马鱼胚胎发育中的作用

发布时间：2018-05-30 02:20

本文选题：组蛋白赖氨酸脱甲基酶 + ES细胞　；参考：《复旦大学》2009年博士论文

【摘要】：组蛋白赖氨酸脱甲基酶Lsd1 (Lysine specific demethylase 1)催化H3K4me1/2或H3K9mel/2脱甲基化从而调节染色质结构与基因转录。随着表观遗传学的飞速发展,人们已经认识到表观遗传调控在发育分化与疾病发生中起着重要的作用。组蛋白赖氨酸甲基化与脱甲基化介导的基因转录调控是发育分化过程中表观遗传调节的重要部分,然而Lsd1在其中的具体作用尚不清楚。因此,我们分别以小鼠胚胎干细胞(embryonic stem cells, ES cells)与斑马鱼为体外和体内模型,采用RNA干扰、Morpholino反义核酸、化学抑制剂处理、基因芯片、染色质免疫沉淀等实验技术研究Lsdl在发育分化中的作用。我们采用慢病毒介导的shRNA在小鼠ES细胞中成功的下调了Lsdl的表达,结果显示：①下调Lsd1表达不影响ES细胞的自我更新：细胞形态以及多能性标志基因Oct4、Nanog、Sox2与Rexl的表达水平未发生显著改变。②在EB(embryonic body,拟胚体)分化过程中加入Lsd1的H3K4mel/2脱甲基酶活性抑制剂TCP (Tranylcypromine,反式环苯丙胺),红细胞标志基因Globin等的表达被显著抑制,但内皮细胞标志基因VE-Cadherin等的表达没有变化,胚胎早期发育相关基因的表达也没有明显变化。③下调Lsdl表达显著抑制EB分化过程的中胚层细胞发育分化：早期中内胚层标志基因Brachyury的表达被延迟,造血/内皮祖细胞标志基因Flkl、造血祖细胞标志基因Scl、红系祖细胞标志基因Gatal及红细胞标志基因Globin等的表达被显著抑制。④染色质免疫沉淀检测显示EB分化过程中Lsd1可以结合在cKit基因的启动子区域,TCP处理引起EB10天时造血相关基因启动子区域H3K4me2水平上升。⑤基因芯片检测结果显示下调Lsd1表达导致中胚层发育延迟,早期中胚层标志基因在EB6天仍然高表达。我们的研究结果证明Lsd1调节胚胎早期的发育分化,其中对原始造血分化的调节作用依赖于H3K4mel/2脱甲基酶活性。以斑马鱼为模型的实验结果显示：①TCP处理的斑马鱼胚胎在30 hpf时出现“无血(Bloodless)"表型,而用帕吉林(Pargyline)和丙酰苄胺异烟肼(Nialamide)处理则不引起类似表型。②进一步检测显示TCP抑制原始红细胞生成,但对血管发生没有显著影响；TCP处理的Tg (gatal:EGFP)斑马鱼胚胎在30 hpf时血液循环中EGFP阳性细胞数目减少,并且胚胎globin基因表达减少。③克隆表达纯化了zLsd1的C端161-867a.a.(含SWIRM与FAD结构域,GST-zLsd1161-867aa),脱甲基酶活性测试显示体外重组表达的GST-zLsdl 161-867a.a具有H3K4me2脱甲基酶活性,并且TCP可以抑制体外重组表达的GST-zLsd1161-867aa对H3K4me2的脱甲基酶活性。④注射干扰zLsd1 mRNA正常剪接的反义核酸(lsd1-MO)也导致斑马鱼胚胎在30 hpf时出现“无血”表型、血液循环中gata1:EGFP阳性细胞数目减少,胚胎globin基因表达减少。⑤基因芯片检测结果显示注射lsd1-MO的斑马鱼胚胎在24hpf时胚胎globin基因的表达均被抑制,其它一些红细胞相关标志基因的表达也被抑制。我们在两种生物学模型中的研究结果均表明Lsd1在胚胎发育与细胞分化中发挥着重要的作用：Lsdl调节中胚层发育分化及原始红细胞生成。Lsd1对早期中胚层发育分化的作用不依赖于H3K4me1/2脱甲基酶活性,但对原始红细胞生成的调节作用主要依赖于H3K4me1/2脱甲基酶活性。Lsd1调节不同阶段发育分化的具体机制以及在血液等相关疾病中作用还有待进一步研究。
[Abstract]:Histone lysine demethanase Lsd1 (Lysine specific demethylase 1) catalyzes the demethylation of H3K4me1/2 or H3K9mel/2 to regulate chromatin structure and gene transcription. With the rapid development of epigenetics, it has been recognized that epigenetic regulation plays an important role in the development of differentiation and disease. Histone lysine Methylation and demethylation mediated gene transcription regulation is an important part of epigenetic regulation during developmental differentiation. However, the specific role of Lsd1 is not clear. Therefore, we use mouse embryonic stem cells (embryonic stem cells, ES cells) and zebrafish as in vitro and in vivo models, using RNA interference, Morpholino reaction. The role of Lsdl in the development and differentiation was studied by using nucleic acid, chemical inhibitor, gene chip and chromatin immunoprecipitation.
The expression of Lsdl was downregulated by lentivirus mediated shRNA in mouse ES cells. The results showed that: (1) downregulation of Lsd1 expression did not affect the self renewal of ES cells: cell morphology and pluripotent marker gene Oct4, the expression level of Nanog, Sox2 and Rexl had not changed significantly. 2. In EB (embryonic body, the embryoid body) The expression of Lsd1 H3K4mel/2 demethylase activity inhibitor TCP (Tranylcypromine, trans ring amphetamine) and erythrocyte marker gene Globin were significantly inhibited during the process, but the expression of the endothelial cell marker gene VE-Cadherin and so on did not change, and the expression of the early embryonic development related genes had not changed obviously. (3) down regulated Lsdl expression. The development and differentiation of mesoderm cells in the EB differentiation process: the expression of the early mesoderm marker gene Brachyury was delayed, the hematopoietic / endothelial progenitor marker gene Flkl, the hematopoietic progenitor marker gene Scl, the erythroid progenitor cell marker gene Gatal and the erythrocyte marker gene Globin were significantly suppressed. The detection showed that Lsd1 could be combined with the promoter region of the cKit gene during the differentiation of EB. TCP treatment caused the increase of H3K4me2 level in the promoter region of hematopoietic related genes at EB10 days. 5. The results of gene chip detection showed that the down-regulation of Lsd1 expression led to the delayed development of mesoderm, and the early mesoderm gene was still highly expressed in EB6 days. The results showed that Lsd1 regulates the differentiation and development of early embryos, and its regulation on primitive hematopoietic differentiation depends on the activity of H3K4mel/2 demethylation.
The experimental results of zebrafish model showed that: (1) TCP treated zebrafish embryos had "Bloodless" phenotypes at 30 HPF and no similar phenotype was caused by the treatment of Bloodless (Pargyline) and propanylbenzamine isoniazid (Nialamide). 2. Further tests showed that TCP inhibited the formation of original red blood cells, but there was no significant effect on blood vessels. The number of EGFP positive cells in the blood circulation of Tg (gatal:EGFP) zebrafish treated by TCP decreased and the expression of globin gene decreased. (3) the clone expressed and purified 161-867a.a. (SWIRM and FAD domain, GST-zLsd1161-867aa) and purified the C end of zLsd1, and the demethylation activity test showed that the recombinant expression of zLsd1 was 16. 1-867a.a has H3K4me2 demethylation activity, and TCP can inhibit the reorganized GST-zLsd1161-867aa demethase activity of H3K4me2 in vitro. (4) injecting zLsd1 mRNA normal splicing antisense nucleic acid (lsd1-MO) also leads to the appearance of "blood free" phenotype in zebrafish embryos at 30 HPF, and the number of gata1:EGFP positive cells in blood circulation. The expression of globin gene decreased. 5. The results of gene chip detection showed that the expression of globin gene in the embryo of zebrafish injection of lsd1-MO was suppressed at 24hpf, and the expression of some other erythrocyte related genes was also suppressed.
Our results in two biological models show that Lsd1 plays an important role in embryonic development and cell differentiation: Lsdl regulates the development and differentiation of mesoderm and the effect of.Lsd1 on the development and differentiation of the early mesoderm does not depend on the activity of the H3K4me1/2 demethase, but the regulation of the formation of the original erythrocyte The specific mechanisms that mainly rely on the H3K4me1/2 demethylase activity.Lsd1 to regulate the development and differentiation of different stages and the role in the blood related diseases need to be further studied.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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