Isolation and Characterization of Yeast rDNA Chromatin

描述(申请人提供)：基因是如何开启和关闭的？如果DNA序列保持不变，转录的变化是由DNA相关因子的差异引起的。因此，了解DNA特定区域内染色质组成的变化对于了解基因表达至关重要。这一建议旨在通过利用质谱仪检测酿酒酵母中核糖体DNA(RDNA)染色质组成与rDNA转录的关系来更好地了解染色质动态在基因表达中的作用。这个项目有三个具体目标。一是从酵母细胞中提纯天然rDNA染色质。其次，结合蛋白的特性[和位置]以及组蛋白修饰的性质将使用质谱学来确定。第三，将确定活跃转录的和沉默的rDNA在染色质组成和组蛋白修饰方面的差异。未来的方向包括将这项技术从酵母扩展到哺乳动物细胞。关键位点的染色质组成可以通过发育漏出、环境变化或在肿瘤发生过程中进行追踪。我的最终目标是完整地描述有机体的“染色体组”：与每个DNA序列相关的所有蛋白质的身份、数量和位置，以及它们在发育、衰老和疾病过程中的修饰状态。

DESCRIPTION (provided by applicant): How do genes turn on and off? If DNA sequence remains unchanged, changes in transcription are caused by differences in DNA-associated factors. So understanding chromatin composition changes within particular regions of DNA is vital to understanding gene expression. This proposal aims to better understand the role of chromatin dynamics in gene expression by examining how ribosomal DNA (rDNA) chromatin composition relates to rDNA transcription in the yeast Saccharomyces cerevisiae using mass spectrometry. This project has three specific aims. The first is to purify native rDNA chromatin from yeast cells. Second, the identity [and locations] of bound proteins and nature of histone modifications will be determined using mass spectrometry. Third, differences in chromatin composition and histone modifications between actively transcribed and silent rDNA will be determined. Future directions include extending the technology from yeast to mammalian cells. Chromatin composition at crucial loci could be followed through developmental transudations, environmental changes, or during oncogenesis. My ultimate goal is the complete characterization of an organism's "chromatome:" the identity, quantity, and location of all proteins associated with every DNA sequence and their modification states through development, aging and disease.