原沉默子及染色质重构蛋白对基因沉默和异染色质结构的作用

发布时间：2018-05-06 22:10

本文选题：基因沉默 + 沉默子　；参考：《吉林大学》2011年博士论文

【摘要】：酿酒酵母染色质的大部分端粒附近,rDNA区域以及HML和HMR区域基因是沉默不表达的,形成了类似高等真核生物的异染色质结构。沉默子HML-E和HML-I对其第三号染色质的HML区域基因沉默起着决定作用,它们由复制起点识别复合物(Origin Recognition Complex, ORC)结合位点, (repressor activator Protein, Raplp)结合位点和自主复制序列(autonomously replicating sequence binding factor, Ab.f1p)结合位点中的三个或者两个结合位点所组成,这些结合位点被称为原沉默子(protosilencer)。沉默子通过多种蛋白募集沉默信息(silent information regulator, Sir)蛋白到它上面,进而沿着染色质纤维传递,形成基因沉默的异染色质区域。但组成沉默子的原沉默子单独情况下对基因沉默的作用尚未研究清楚,此外常染色质与异染色质之间的转化需要染色质的重构,但任意一种Sir蛋白都没有染色质重构活性,而染色质重构因子例如Fun30, Snf2, IswI等对基因沉默起一定作用,但是染色质重构因子在基因沉默以及异染色质形成过程中的具体作用尚未研究清楚。为研究原沉默子对基因沉默的作用,本研究采用带有报告基因URA3的不同原沉默子替代HML区域HML-I沉默子,比较URA3的基因表达的差别,发现原沉默子单独情况下没有沉默作用,但与HML-E沉默子共同作用下能在两者之间形成一定的基因沉默；为研究原沉默子拷贝数对基因沉默的影响,本研究利用带有报告基因URA3的不同拷贝数的原沉默子Abflp结合位点替代HML-I沉默子,发现增加拷贝数能增强基因沉默作用；为研究原沉默子在不同位置对基因沉默的影响,本研究利用带有报告基因URA3的原沉默子Raplp结合位点在不同位置的序列替代HML-I沉默子,发现Raplp在本身原来的位置有一定的基因沉默作用,在其他位置基本没有基因沉默作用。为研究原沉默子基因沉默产生差异的原因,通过DNA拓扑结构法研究了不同条件下原沉默子对该区域染色质DNA螺旋程度产生的影响,发现发现负超螺旋DNA的比例Abflp结合位点最高,ORC次之,Rap1结合位点最弱；为研究它们对对染色质结构产生的影响,通过不完全MNase降解结合Southern blot法发现不同的原沉默子与沉默子相互作用下原沉默子附近的核小体结构发生较大变化,但沉默子附近的染色质结构没有发生变化,通过染色质结构变化上不同推测,可能是沉默子与原沉默子之间相互作用会随着它们种类,数目,位置的不同而不同,从而导致形成的基因沉默上存在差别。为研究重构蛋白在异染色质重排中的具体作用,本研究敲除了酵母的IswIp, Isw2p, Chdlp, Ino80p, Rad54p, Fun30等基因。发现敲除Isw2p, Chdlp, Ino80p, Rad54p基因对酵母的基因沉默没有影响,而敲除IswIp, Fun30基因会酵母的基因沉默产生影响；通过拓扑结构法以及MNase不完全降解结合Southern blot法发现敲除Fun30对异染色质的形成影响很大而对异染色质的维持影响不大,而敲除IswI对异染色质的形成影响不大但对异染色质的稳定维持影响很大。本研究创新之处：1)不同的原沉默子对插入的外源基因有不同的沉默作用,对异染色质核小体的排列以及染色质DNA螺旋有不同的影响；2)增加原沉默子Abfl结合位点拷贝数能增强基因沉默作用：3)改变原沉默子的排列也会影响基因沉默；4)染色质重构因子Fun30和IswI这两种蛋白对基因沉默起增强作用；5)Fun30对异染色质的形成起重要作用而对异染色质的稳定维持起很小作用,而IswI对异染色质的形成起很小作用,而对异染色质的稳定维持起重要作用。
[Abstract]:Near the most telomere of Saccharomyces cerevisiae, the rDNA region and the HML and HMR region genes are silent and non expressed, forming a heterochromatin similar to the higher eukaryotes. The silencing HML-E and HML-I play a decisive role in the HML region gene silencing of its third chromatin, which are identified by the replication starting point (Origin Recogn). Ition Complex, ORC) binding sites (repressor activator Protein, Raplp) binding sites and three or two binding sites in the binding site (autonomously replicating sequence binding factor). These binding sites are called the original silencers. The silencers pass through a variety of eggs. The white recruitment of silent information regulator (Sir) protein is above it, and then transmitted along the chromatin fiber to form a gene silenced heterochromatin region. However, the role of the original silencer, which is composed of the silent child, has not been clearly studied, and the transformation between the outer chromatin and heterochromatin needs to be dyed. However, any kind of Sir protein has no chromatin remodeling activity, but chromatin remodeling factors such as Fun30, Snf2, and IswI play a role in gene silencing, but the specific role of chromatin remodeling factor in the process of gene silencing and heterochromatin has not been clearly studied.
In order to study the effect of the original silencer on gene silencing, this study uses a different original silencer with a reporter gene URA3 to replace the HML-I silencer in the HML region, and compares the difference in the gene expression of the URA3. It is found that the original silencer has no silence in a single case, but it can form a certain gene sink between the two and the HML-E silencers. In order to study the effect of the original silencing number on gene silencing, this study uses the Abflp binding site of the original silencer with different copies of the reporter gene URA3 to replace the HML-I silencer. It is found that the increase of the number of copies can enhance the gene silencing effect. The original silencer Raplp binding site of the reporter gene URA3 replaced the HML-I silencer in different locations. It was found that Raplp had a certain gene silencing effect in its original position, and there was no gene silencing in other locations. The reason for the difference of the silence of the original silencing subgene was studied by the DNA topological structure method. Under the same condition, the effect of the original silencer on the degree of helicity of chromatin DNA in the region was found. It was found that the proportion of Abflp binding sites in the negative super spiral DNA was the highest, ORC time and the Rap1 binding site were the weakest, and the effect of them on the chromatin structure was studied, and the different original silencers were found by the incomplete MNase reduction and Southern blot method. The nucleosome structure in the vicinity of the original silencer changes greatly with the interaction of the silencer, but the chromatin structure near the silencer does not change. It is presumed that the interaction between the silencer and the original silencer may be different with the species, the number and the position of the original silencers. There is a difference in the formation of gene silencing.
In order to study the specific role of recombinant protein in heterochromatin rearrangement, this study knocked out the genes of yeast IswIp, Isw2p, Chdlp, Ino80p, Rad54p, Fun30. It was found that the knockout of Isw2p, Chdlp, Ino80p, Rad54p genes had no effect on the gene silencing of yeast, and the Fun30 gene would affect the gene silencing of yeast; Papping structure method and MNase incomplete degradation combined with Southern blot method found that knockout Fun30 has a great influence on the formation of heterochromatin, but it has little effect on the maintenance of heterochromatin, but the knock off of IswI has little effect on the formation of heterochromatin but has a great influence on the stability of heterochromatin.
The innovation of this study: 1) different original silencers have different silencing effects on the inserted foreign genes, and have different effects on the arrangement of heterochromatin nucleosomes and chromatin DNA helix; 2) increasing the copy number of the binding site of the original silencer can enhance the gene silencing effect: 3) change the arrangement of the original silencer and affect the gene sink. 4) two proteins, chromatin remodeling factors Fun30 and IswI, enhance the gene silencing; 5) Fun30 plays an important role in the formation of heterochromatin and has a small effect on the stability of heterochromatin, while IswI plays a small role in the formation of heterochromatin, and it plays an important role in maintaining the stability of heterochromatin.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

【共引文献】