LARGE SCALE CHROMATIN STRUCTURE/FUNCTION

在高等真核细胞的染色体中，DNA被折叠成多个层次的组织。这些加在一起产生的DNA比10,000：1的线性凝聚更大。最高水平的染色质折叠，包括30 nm染色质纤维的折叠，占高达250：1的包装比，涉及整个转录和复制功能域的组织，它们在有丝分裂和间期染色单体中的紧凑，以及它们在间期核中的结构。这种大规模的染色质结构是我们研究的重点。我们的长期目标是确定大规模染色质结构和染色体结构背后的基本折叠基序，与转录和复制相关的特定构象变化的功能意义，以及调节这些构象转变的潜在机制。我们的具体目标是回答以下问题：1)有丝分裂细胞周期中大规模染色质凝聚/解凝聚的结构基序是什么？2)有丝分裂染色体形成过程中染色质大规模凝聚的哪些步骤取决于SMC蛋白的功能？3)大规模染色质结构和核内定位的变化与DNA复制启动有关？4)浓缩和延伸的间期大规模染色体结构对DNA序列的要求是什么？此外，我们还将探索几种实验方法，以测试特定蛋白质在维持间期染色体内特殊的大规模染色质结构中的作用，并寻找参与大规模染色质组织和核结构的新蛋白质。这项拟议的研究将提供大规模染色质和染色体组织背后的折叠基序的基本描述。我们未来的工作方向将集中在将大规模染色质组织的结构模型与潜在的生物化学和分子生物学相结合。我们特别感兴趣的是探索转录、复制以及大规模染色质和核结构之间的机制联系。从这项研究中获得的知识应该为转录调控、改进基因治疗载体的设计和稳定的转基因表达提供有价值的见解。

Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization. Together these yield greater than a 10,000:1 linear condensation of DNA. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 250:1 packing ratio and involve the organization of entire transcription and replication functional domains, their compaction within mitotic and interphase chromatids, and their architecture within interphase nuclei. Essentially uncharacterized at this time, it is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure and chromosome architecture, the functional significance of specific conformational changes associated with transcription and replication, and the underlying mechanisms which regulate these conformational transitions. Our specific aims are to answer the following questions: 1) What structural motifs underlie large-scale chromatin condensation/decondensation during the mitotic cell cycle? 2) Which steps in large-scale chromatin condensation during formation of mitotic chromosomes are dependent on SMC protein function? 3) What changes in large scale chromatin structure and intranuclear positioning are associated with DNA replication initiation? 4) What are the DNA sequence requirements for condensed versus extended interphase large-scale chromosome structures? In addition we will explore several experimental approaches to test the role of specific proteins in maintaining specialized large-scale chromatin structures within interphase chromosomes and to search for new proteins involved in large- scale chromatin organization and nuclear architecture. This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on integrating structural models of large-scale chromatin organization with the underlying biochemistry and molecular biology. In particular we are interested in exploring the mechanistic links between transcription, replication, and large- scale chromatin and nuclear structure. Knowledge gained from this research should provide valuable insight into regulation of transcription and improved design of gene therapy vectors and stable transgene expression.