Genome-Wide Mapping of Chromosomal Proteins in Drosophilia

描述（由申请人提供）：完整或近乎完整的真核生物基因组序列的产生产生了对基因组编码潜力和进化的爆炸性见解。然而，我们对涉及DMA的关键过程是如何调控的信息较少，包括转录、复制、修复和染色体分离。单靠DMA序列不能揭示基因组调控和遗传的机制，因为这些过程并不作用于“裸”DMA。在细胞中，DMA被包装成染色质，染色质的组成和组织调节着DMA序列的可及性和功能。染色质显示出越来越复杂的组织和组成水平，从基本的核小体单位开始，通过更高的顺序结构。染色质组织对于利用存储在基因组中的信息至关重要；不能准确地靶向或维持染色体蛋白质和染色质成分会导致基因表达和染色体行为的异常模式，并与许多人类疾病有关，尤其是癌症。我们提议通过确定果蝇基因组中125个染色体蛋白和组蛋白修饰的位置来参与MODENCODE项目。所研究的蛋白质和修饰涉及基本的染色体功能，如DNA复制、基因表达、基因沉默和遗传。我们将使用本项目生成并验证的商业化抗体进行染色质免疫沉淀（ChIP），分离和标记沉淀的DNA，并将探针应用于基因组平铺阵列。通过扫描杂交阵列产生的数据将通过统计方法进行分析，并且阵列数据将通过在细胞和动物中的独立分析来验证。我们将首先使用来自三个细胞系和两个胚胎阶段的染色质进行定位分析，然后将蛋白质子集的分析扩展到四个额外的动物组织/阶段。然后，我们将在蛋白质“景观”数据集之间进行各种比较，包括分析修饰和染色体蛋白质的组合模式，组织特异性差异以及参与相同表观遗传途径的蛋白质之间的相互作用。最后，所有经过验证的数据和分析将提供给ENCODE项目的成员和科学界。该项目的成功完成将为果蝇基因组中染色质组分的分布提供基本信息，为今后的功能研究奠定基础。此外，这些数据和分析极有可能为理解染色质在人类细胞和疾病中的作用提供关键信息。

DESCRIPTION (provided by applicant): The generation of complete or nearly complete eukaryotic genome sequences has produced an explosion of insights into the coding potential and evolution of genomes. However, we have less information about how key processes involving DMA are regulated, including transcription, replication, repair, and chromosome segregation. DMA sequence alone cannot be expected to reveal the mechanisms involved in genome regulation and inheritance, because these processes do not act on 'naked' DMA. In the context of the cell, DMA is packaged as chromatin, whose composition and organization regulate the accessibility and function of DMA sequences. Chromatin displays increasingly complex levels of organization and composition, starting with the basic nucleosome unit and progressing through higher order structures. Chromatin organization is critical for utilizing information stored in the genome; failure to accurately target or maintain chromosomal proteins and chromatin components results in aberrant patterns of gene expression and chromosome behavior, and is associated with many human diseases, most notably cancer. We propose to participate in the MODENCODE project by determining the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) with antibodies obtained commercially and generated and validated by this project, isolate and label the precipitated DNA, and apply the probes to genomic tiling arrays. Data generated by scanning the hybridized arrays will be analyzed by statistical methods, and the array data will be validated by independent analyses in cells and animals. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. We will then perform a variety of comparisons between protein 'landscape' data sets, including analyses of combinatorial patterns of modifications and chromosomal proteins, tissue-specific differences, and interactions among proteins involved in the same epigenetic pathways. Finally, all validated data and analyses will be made available to members of the ENCODE project and scientific community. Successful completion of this project will provide basic information about the distributions of chromatin components across the Drosophila genome sequence, which will serve as a foundation for future functional studies. In addition, the data and analysis are highly likely to provide information critical to understanding the roles of chromatin in human cells and diseases.