原沉默子对基因沉默和异染色质结构作用

Chromatin is divided into different regions，which are loosely structured euchromatin and highly condensed chromatin heterochromatin. Gene expression is normal in euchromatin， but gene expression is silent in heterochromatin which is also known as silent chromatin.Heterochromatin in Saccharomyces cerevisiae exists at HML and HMR loci as well as subtelomeric region. Like its metazoan counterpart, Saccharomyces cerevisiae heterochromatin consists of highly like its metazoan counterpart, its heterochromatin consists of highly ordered and stably positioned nucleosomes.In Saccharomyces cerevisiae,transcriptionally silent heterochromatin at the HML and HMR loci is established by silencers that recruit and promote the propagation of the SIR silencing complex along chromatin.Siencers are composed of various combinations of the two or three of the binding sites for origin recognition complex (ORC), autonomously replicating sequence binding factor1 (Abf1) and repressoractivatorprotein1 (Rap 1) proteins of that are believed to play similar and redundant roles in gene silencing and fromation of heterochromatin.Individual ORC, Abf1 and Rap1 sites alone are not able to promote silencing, but have the potential of enhancing silencing by a distant silencer, and have therefore been referred to as protosilencers.However,the mechanisms underlying the function of protosilencers have not been resolved.In this study,we exmained the functions of the ORC, Abf1,Rap1 sites from the HMR-E silencer as protosilencers. We found that protosilencer alone is unable to produce gene silencing; with the help of HML-E silencer, protosilencer Abf1p and ORC binding sites can produce gene silencing on on its both sides while Rap1 binding site only have gene silencing on its right side, these results indicate that protosilencer play auxiliary role on gene silencing, and Rap1 binding site not only play an auxiliary role on gene silencing, but also possesses boundary activity. We also found that increasing the copy number of Abf1 binding sites can enhance its role of gene silencing with help of HML-E silencer, and surprisingly two copies of the Abf1 binding site alone exhibits gene silencing without HML-E silencer , but the reasons for the change still needs further study; we also found that degree of gene silencing is different in different locations, and only has gene silencing effect in its original position, and the possible explanation for the difference maybe because of the sequence around it could enhance its role in gene silecing.So we plan to use DNA topology assay,Mnase digestion and indirect end-labeling, Chip and 3C to study chromatin supercoil, nuclesome array,histone modification and the relationship of cis-acting DNA element for better studingy the underly mechanisms.

真核生物染色质分为常染色质和异染色质，异染色区域基因沉默是目前研究的热点。酿酒酵母遗传背景清楚，便于进行基因沉默的研究，迄今为止HM区域基因沉默研究最多，该区域两侧的沉默子形成，延伸，维持基因沉默。沉默子由原沉默子ORC，Abf1，Rap1结合位点中两个或三个组成。本研究前期工作表明，原沉默子单独存在虽无法形成基因沉默，但是与沉默子相互作用下，Abf1和ORC结合位点能够在其两侧形成基因沉默，而Rap1结合位点只在与沉默子相互作用侧形成基因沉默，而在其另外一侧无法形成基因沉默，改变Rap1位置基因沉默消失，Abf1结合位点单独存在2或3个拷贝数均形成基因沉默。本研究计划分别采用DNA拓扑结构法、MNase不完全降解结合Southern blot法、CHIP、3C对染色质螺旋度、核小体排列、组蛋白修饰、空间距离等染色质结构的角度对基因沉默的机制进行研究并解释。

在酿酒酵母中，在HLM和HMR区域形成了基因沉默的异染色质，该区域基因沉默由沉默子起始并形成。沉默子由ORC（origin recognition complex for DNA replication， ORC），Abf1，Rap结合位点中的两个或三个多种方式组合而成。单独的ORC，Abf1，Rap1结合位点不能形成基因沉默，但能辅助增强附近的沉默子的基因沉默，因而这些位点被称为原沉默子。原沉默子发挥功能的机制目前还不清楚。我们通过研究组成HMR-E沉默子的ORC，Abf1，Rap1结合位点的功能来研究原沉默子的功能。我们发现原沉默子ORC和Abf1结合位点能辅助增强HML-E沉默子的形成异染色质的功能，但是Rap1结合位点却不能。然而原沉默子ORC或Abf1与HML-E沉默子形成的异染色质稳定性低于HML-E与HMR-E这两个沉默子形成的，增加Abf1的拷贝数能够增加形成的异染色质的稳定性。原沉默ORC和Abf1结合位点，在缺乏SIR蛋白的情况下不能改变染色质核小体的排列。我们还发现增加原沉默子的ORC和Abf1结合位点与沉默子HML-E之间的距离，基因沉默的模式不会发生改变，染色质构象捕获结果表明这两个原沉默子是通过与沉默子空间上相互接触来增强两者之间的基因沉默。我们也研究了包含原沉默子ORC结合位点和Abf1结合位点的自主复制序列（autonomously replicating sequence， ARS）1的基因沉默功能。我们发现ARS插入到HML区域能够增强异染色质的稳定性，并且以细胞周期S期依赖性的方式形成了与异染色质相似的染色质结构。本项目的研究表明原沉默子能够辅助形成和维持异染色质结构，其增强沉默子基因沉默机制可能是通过与沉默子在空间上相互接近来实现的。