Genome-wide profiling of histone variants in Drosophila and Caenorhabditis

描述(由申请人提供)：我们建议在带注释的黑腹果蝇和秀丽线虫基因组中以高分辨率绘制主要的核心组蛋白变体图谱。这些图谱将提供一个基于染色质的框架，用于解释表观遗传信息和其他人确定的基因表达模式。为了实现这一点，我们将使用我们已经成功应用于基因组规模染色质亲和纯化的生物素标记系统和标准的微阵列读出平台。在黑腹毛虫和线虫中，我们都将描述普遍复制无关的组蛋白3变异体H3.3，以及控制复制耦合变异体H3。这些数据将提供两种模式生物体中组蛋白动态的高分辨率全基因组图谱。H3.3模式与全基因组活跃的组蛋白修饰模式以及同源异型基因簇上的DNasel超敏感位点的密切对应表明，我们的全基因组图谱可用于验证旨在定位这些其他基因组特征的努力。在黑腹毛虫和线虫中，我们还将描述与各种不同生物体的表观遗传过程有关的h2AZ变体。果蝇H_2AZ作为H_2AX变异体具有独特的双重作用，称为H_2AV，我们的初步研究表明H_2AX的模式与H_(3.3)的模式不同，从而在全基因组范围内提供了不同的组蛋白动态的表观遗传学特征。为了扩大我们对这两类组蛋白变体功能作用的理解，我们将使用RNAi在果蝇细胞系和整个线虫不同发育阶段中下调关键染色质调节因子的功能。敲除变体及其组装机器后的转录图谱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体图谱应有助于识别靶基因。与其他类型的全基因组信息一起，我们的组蛋白变异图可能有助于更全面地理解这两种模式生物基因组中的表观遗传调控元件。鉴于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发挥着相似的作用，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.