Chromosome Structure and Gene Expression

描述(申请人提供)：所有生物的染色体都经历转录、DNA复制/修复和分离到子代细胞的过程。染色质是真核生物中染色体DNA和蛋白质的复合体，对这些基本过程有着深远的影响，染色质调节缺陷与包括癌症在内的多种人类疾病有关。染色质纤维由核小体的重复亚单位组成，被用作组装复杂的高阶结构的基础，涉及额外的染色体凝聚、环状和折叠。这项研究的目的是利用生物化学和遗传学的结合来帮助理解单细胞微生物酿酒酵母中高阶染色质的组装。重点是在一个称为毛发的位置形成“沉默的染色质”，它是高阶染色质的结构、功能和可遗传性质的范例。HMR沉默包括-4kb的染色体DNA，由一种名为Silencer的小DNA元件控制，该元件通过直接与Silencer结合蛋白(如起源识别复合体(ORC))发挥作用，后者又通过蛋白质-蛋白质相互作用招募专门的沉默蛋白，如Sirlp。这种沉默蛋白复合体“核”了沉默的染色质的形成，包括特殊的非组蛋白蛋白的结合，如Sir3p与染色质纤维的结合。特定的Sirlp-ORC相互作用是形成沉默蛋白复合体的中心。ORC还在更一般和更基本的复制启动过程中发挥作用，这些复制起始于数百个贯穿整个基因组的“通用”(非沉默)复制起始点。因此，某些机制必须将功能性Sirlp-ORC相互作用限制在消音器上。我们推测，ORC-DNA相互作用和额外的染色质结合蛋白确保Sirlp和ORC之间稳定的物理相互作用只发生在消音器上。此外，我们推测HMR内的ORC-DNA相互作用抑制了ORC在复制启动中的功能。最后，我们假设Sir3p在HMR与核小体结合，并直接有助于将HMR染色质纤维“重塑”成更大的“高阶”染色质区域。

DESCRIPTION (provided by applicant): The chromosomes of all organisms are subject to the processes of transcription, DNA replication/repair and segregation to daughter cells. Chromatin, the complex of chromosomal DNA and proteins in eukaryotes, has a profound impact on these fundamental processes, and defects in chromatin regulation are associated with a variety of human diseases including cancer. The chromatin fiber, which consists of repeating subunits of nucleosomes, is used as the foundation for the assembly of complex higher-order structures involving additional levels of condensation, looping and folding of chromosomes. The goal of this research is to contribute to an understanding of the assembly of higher-order chromatin using a combination of biochemistry and genetics in the single-celled microbe Saccharomyces cerevisiae. The focus is the formation of "silenced chromatin" at a locus called HAIR that serves as a paradigm for the structure, function, and heritable nature of higher-order chromatin. HMR silencing encompasses -4 kb of chromosomal DNA and is controlled by a small DNA element called a silencer that functions by binding directly to silencer-binding proteins such as the Origin Recognition Complex (ORC) that in turn recruit specialized silencing proteins, such as Sirlp, through protein-protein interactions. This silencer-protein-complex "nucleates" the formation of silenced chromatin, which includes the binding of specialized non-histone proteins, such as Sir3p to the chromatin fiber. A specific Sirlp-ORC interaction is central to formation of the silencer-protein-complex. ORC also functions in the more general and essential process of replication initiation at the hundreds of "generic" (non-silencer) replication origins through out the genome. Thus some mechanism must confine a functional Sirlp-ORC interaction to silencers. We postulate that ORC-DNA interactions and additional chromatin-binding proteins insure that a stable physical interaction between Sirlp and ORC occurs only at silencers. In addition, we postulate that ORC-DNA interactions within HMR suppress ORC's function in replication initiation. Lastly, we postulate that the Sir3p binds to nucleosomes at HMR and contributes directly to "remodeling" the HMR chromatin fiber into a larger domain of "higher-order" chromatin.