生殖细胞特异性基因的表达受甲基化和转录因子的双重调控
发布时间：2018-07-15 22:34
【摘要】：在体内原始生殖细胞(PGC, primordial germ cell)的数目是极其有限的,最终约26,000个,且不易分离得到；目前已知在PGC形成初期特异性表达Blimp1、中期Stella和Nanos3,后期表达Vasa、Dazl及减数分裂时期表达Nanos2和Stra8外,其他已知的PGC特异性标志基因很少；另外,目前人类对PGC形成的机理尚不清楚。基于上述原因,我们利用传统的EB (embryonic body)体外分化体系来模拟PGC的分化形成,通过基因的筛选希望能找到促进ES (embryonic stem cell)细胞分化形成PGC的基因,并进而发现PGC细胞新的标志性基因,从而对PGC的形成机理有更深入的了解。 本论文包括两部分内容：第一部分是能促进胚胎干细胞向原始生殖细胞分化的基因的筛选。通过传统的EB分化体系、SSEA1染色和AP染色,检测了基因Sohlh1、Sohlh2、 Fgd1和Fkbp3对原始生殖细胞形成的影响。发现基因Sohlh1对PGC的形成可能有促进作用,基因Sohlh2、Fgdl和Fkbp3对PGC的形成无明显影响。第二部分内容是生殖细胞特异性基因的表达受转录因子和DNA甲基化的双重调节。基于本实验室的其他研究发现生殖细胞特异性基因As1对ES向原始生殖细胞的分化起促进作用,在研究其作用机理的过程中发现转录因子Nrf1(Nuclear respiratory factor1)结合在它的启动子上,对其表达有一定的调节作用,通过荧光素酶报告基因技术(luciferase assay)的进一步证明了Nrf1对Asz1的表达起到促进作用,而且Nrfl的结合位点富含CG,结果表明Nrf1的结合受到DNA甲基化的影响。我们广泛分析了一些生殖细胞特异性基因的启动子,发现基因Ddx25、Lin28上都有Nrf1的结合位点,且基因Ddx25的启动子上也富含CG。通过荧光素酶报告基因技术,我们证明了Nrf1能够促进基因Ddx25和Lin28的表达,当Ddx25被体外甲基化处理后,Nrf1的促进作用消失。另外,我们发现许多生殖细胞特异性的基因的启动子上含有转录因子Oct1的结合位点,并通过荧光素酶报告基因技术试验证明了Oct1能够促进基因Vasa的表达。我们认为Oct1、Nrf1可能对生殖细胞特异性的基因的表达起着重要的调节作用,而这种调控受到甲基化的双重影响。我们的研究,对生殖细胞特异性基因表达的表观遗传学修饰有一个初步的认识。
[Abstract]:In vivo, the number of primordial germ cells (PGCs, primordial germ cell) is very limited, and it is not easy to isolate. It is known that Blimp1, Stella and Nanos3 are specifically expressed in the early stage of PGC formation, Stella and Nanos3 are expressed in the middle stage, VasaanDazl is expressed in the late stage, and Nanos2 and Stra8 are expressed in meiosis. Other known PGC specific marker genes are few, and the mechanism of PGC formation is still unclear. For the above reasons, we used the traditional EB (embryonic body) differentiation system to simulate the differentiation of es (embryonic stem cell) cells in vitro. Through gene screening, we hope to find the genes that promote the differentiation of es (embryonic stem cell) cells. Furthermore, a new signature gene was found in PGC cells, and the formation mechanism of PGC was further understood. This thesis consists of two parts: the first part is the screening of genes that can promote embryonic stem cells to differentiate into primitive germ cells. The effects of genes Sohlh1, Fgd1 and Fkbp3 on the formation of primordial germ cells were detected by traditional EB differentiation system, SSEA1 staining and AP staining. It was found that the gene Sohlh1 might promote the formation of PGC, while the genes Sohlh2Fgdl and Fkbp3 had no effect on the formation of PGC. The second part is that the expression of germ cell specific genes is regulated by transcription factors and DNA methylation. Based on other studies in our laboratory, the germ cell-specific gene As1 was found to promote the differentiation of es into primordial germ cells, and the transcription factor Nrf1 (Nuclear respiratory factor1) was found to bind to its promoter in the course of studying its mechanism. Through luciferase reporter gene technique, (luciferase assay) further proved that Nrf1 promoted the expression of Asz1, and the binding site of Nrfl was rich in CG. the results showed that the binding of Nrf1 was affected by methylation of Asz1. Some promoters of germ cell-specific genes were extensively analyzed. It was found that Nrf1 binding sites were found in the gene Ddx25hLin28, and the promoter of the gene Ddx25 was also rich in CG. By luciferase reporter gene technique, we have proved that Nrf1 can promote the expression of Ddx25 and Lin28, and the promotion of Nrf1 disappeared when Ddx25 was methylated in vitro. In addition, we found the binding sites of transcription factor Oct1 in the promoters of many germ cell-specific genes. Oct1 can promote the expression of Vasa by luciferase reporter gene technique. We believe that Oct1nrf1 may play an important role in regulating the expression of germ cell-specific genes, which is affected by methylation. We have a preliminary understanding of epigenetic modification of germ cell specific gene expression.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:In vivo, the number of primordial germ cells (PGCs, primordial germ cell) is very limited, and it is not easy to isolate. It is known that Blimp1, Stella and Nanos3 are specifically expressed in the early stage of PGC formation, Stella and Nanos3 are expressed in the middle stage, VasaanDazl is expressed in the late stage, and Nanos2 and Stra8 are expressed in meiosis. Other known PGC specific marker genes are few, and the mechanism of PGC formation is still unclear. For the above reasons, we used the traditional EB (embryonic body) differentiation system to simulate the differentiation of es (embryonic stem cell) cells in vitro. Through gene screening, we hope to find the genes that promote the differentiation of es (embryonic stem cell) cells. Furthermore, a new signature gene was found in PGC cells, and the formation mechanism of PGC was further understood. This thesis consists of two parts: the first part is the screening of genes that can promote embryonic stem cells to differentiate into primitive germ cells. The effects of genes Sohlh1, Fgd1 and Fkbp3 on the formation of primordial germ cells were detected by traditional EB differentiation system, SSEA1 staining and AP staining. It was found that the gene Sohlh1 might promote the formation of PGC, while the genes Sohlh2Fgdl and Fkbp3 had no effect on the formation of PGC. The second part is that the expression of germ cell specific genes is regulated by transcription factors and DNA methylation. Based on other studies in our laboratory, the germ cell-specific gene As1 was found to promote the differentiation of es into primordial germ cells, and the transcription factor Nrf1 (Nuclear respiratory factor1) was found to bind to its promoter in the course of studying its mechanism. Through luciferase reporter gene technique, (luciferase assay) further proved that Nrf1 promoted the expression of Asz1, and the binding site of Nrfl was rich in CG. the results showed that the binding of Nrf1 was affected by methylation of Asz1. Some promoters of germ cell-specific genes were extensively analyzed. It was found that Nrf1 binding sites were found in the gene Ddx25hLin28, and the promoter of the gene Ddx25 was also rich in CG. By luciferase reporter gene technique, we have proved that Nrf1 can promote the expression of Ddx25 and Lin28, and the promotion of Nrf1 disappeared when Ddx25 was methylated in vitro. In addition, we found the binding sites of transcription factor Oct1 in the promoters of many germ cell-specific genes. Oct1 can promote the expression of Vasa by luciferase reporter gene technique. We believe that Oct1nrf1 may play an important role in regulating the expression of germ cell-specific genes, which is affected by methylation. We have a preliminary understanding of epigenetic modification of germ cell specific gene expression.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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