Chromosome Structure and Gene Expression

描述（由申请人提供）：所有生物体的染色体都经历转录、DNA复制/修复和分离成子细胞的过程。染色质是真核生物中染色体DNA和蛋白质的复合物，对这些基本过程具有深远的影响，染色质调节的缺陷与包括癌症在内的各种人类疾病有关。由核小体的重复亚基组成的染色质纤维被用作组装复杂的高阶结构的基础，所述高阶结构涉及染色体的额外水平的凝聚、成环和折叠。本研究的目标是有助于了解高阶染色质的组装使用生物化学和遗传学的组合在单细胞微生物酿酒酵母。重点是在一个称为HAIR的位点形成“沉默的染色质”，该位点作为高阶染色质的结构、功能和遗传性质的范例。HMR沉默包括~ 4kb的染色体DNA，并且由称为沉默子的小DNA元件控制，所述沉默子通过直接结合沉默子结合蛋白如起源识别复合物（ORC）而起作用，所述起源识别复合物（ORC）进而通过蛋白质-蛋白质相互作用募集专门的沉默蛋白如Sirlp。这种沉默蛋白复合物使沉默的染色质的形成“成核”，其包括专门的非组蛋白蛋白质如Sir 3 p与染色质纤维的结合。特异性Sirlp-ORC相互作用对于沉默子-蛋白质-复合物的形成至关重要。ORC还在整个基因组中的数百个“通用”（非沉默者）复制起点处的复制起始的更一般和必要过程中起作用。因此，某些机制必须将功能性Sirlp-ORC相互作用限制于沉默剂。我们假设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仅发生在沉默剂处。此外，我们推测，在HMR内的ORC-DNA相互作用抑制ORC的复制起始功能。最后，我们假设，Sir 3 p结合到核小体在HMR和直接有助于“重塑”的HMR染色质纤维成一个更大的域的“高阶”染色质。

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.