Genome-wide profiling of histone variants in Drosophila and Caenorhabditis

描述（由申请人提供）：我们建议以高分辨率绘制整个标注的黑腹果蝇和秀丽隐杆线虫基因组中的主要核心组蛋白变异。这些图谱将为解释表观遗传信息和他人确定的基因表达模式提供基于染色质的框架。为了实现这一目标，我们将使用生物素标记系统，我们已经成功地应用于基因组规模的染色质亲和纯化以及标准的微阵列读出平台。在D. melanogaster和C. elegans中，我们将分析普遍的与复制无关的组蛋白3变体H3.3，以及控制复制耦合的变体H3。这些数据将提供两种模式生物组蛋白动力学的高分辨率全基因组图谱。H3.3模式与全基因组活性组蛋白修饰模式的密切对应，以及与同源基因簇上的DNasel超敏感位点的密切对应，表明我们的全基因组图谱可以用于验证旨在绘制这些其他基因组特征的努力。在黑胃线虫和秀丽隐杆线虫中，我们还将分析H2AZ变异，该变异与各种不同生物的表观遗传过程有关。果蝇H2AZ作为H2AX变异H2AV发挥着独特的双重作用，我们的初步研究表明H2AV的模式与H3.3不同，从而提供了不同的组蛋白全基因组动力学表观遗传谱。为了扩大我们对这两类组蛋白变体的功能作用的理解，我们将使用RNAi来敲除果蝇细胞系和整个秀丽隐杆线虫不同发育阶段的关键染色质调节因子的功能。敲除变异及其组装机器后的转录谱分析将提供它们在基因调控中所起作用的功能验证，而敲除后的组蛋白变异谱分析将有助于识别目标基因。与其他类型的全基因组信息一起，我们的组蛋白变异图谱可能有助于更全面地了解这两种模式生物基因组中的表观遗传调控元件。考虑到相同的组蛋白变体被认为在基本上所有复杂的真核生物中起着相似的作用，我们的基本方法可以立即应用于人类基因组。

DESCRIPTION (provided by the applicant): We propose to map major core histone variants throughout the annotated Drosophila melanogaster and Caenorhabditis elegans genomes at high resolution. These maps will provide a chromatin-based framework for interpreting epigenetic information and gene expression patterns determined by others. To accomplish this, we will use the biotin-tagging system that we have successfully applied for genome-scale chromatin affinity purification together with a standard microarray readout platform. In both D. melanogaster and C. elegans we will profile the universal replication-independent histone 3 variant, H3.3, as well as the control replication-coupled variant, H3. These data will provide high-resolution genome-wide maps of histone dynamics in both model organisms. The close correspondences of H3.3 patterns to patterns of active histone modifications genome-wide and with DNasel hypersensitive sites at homeotic gene clusters suggests that our genome-wide maps can be used to validate efforts aimed at mapping these other genomic features. In both D. melanogaster and C. elegans we will also profile the H2AZ variant, which has been linked to epigenetic processes in a variety of different organisms. Drosophila H2AZ plays a unique dual role as the H2AX variant, called H2AV, and our preliminary studies indicate that patterns of H2AV differ from those of H3.3, thus providing a different epigenetic profile of histone dynamics genome-wide. To expand our understanding of the functional roles of both classes of histone variants, we will use RNAi to knock down the function of key chromatin regulators in Drosophila cell lines and in whole C. elegans at different developmental stages. Transcriptional profiling after knock-down of the variants and their assembly machines will provide functional validation of roles that they play in gene regulation, and histone variant profiling after knock-down should help to identify target genes. Together with other types of genome-wide information our histone variant maps are likely to contribute to a fuller understanding of epigenetic regulatory elements in the genomes of these two model organisms. Given that the same histone variants are thought to play similar roles in essentially all complex eukaryotes, our basic approach can be immediately applied to the human genome.