Strategies for mapping origins in mammalian genomes

描述(申请人提供)：在典型的体细胞基因组中有50,000个活跃的起始点。由于这种复杂性，分子生物学方法只确定了几个起源，而且只有在付出巨大的成本和努力后才能确定。许多基因在S早期活跃的细胞类型中复制，但在不活跃的细胞类型中复制较晚，这表明在发育过程中起始活动和转录之间存在相互作用。根据对少数已证实的起始点的生化分析，似乎有两种类型：1)低效起始点的宽广区域，和2)高度优先的起始点。我们认为基因组以1kb或更小的间隔排列着潜在起始点的层次结构，其中一个子集已经进化成真正的复制子。任何给定位置的使用都被认为是由局部基因活性和染色质结构来调节的。高质量人类、小鼠和大鼠基因组序列的完成以及微阵列的出现，为对复制起点的分布、结构和调节进行全球分析提供了一个独特的机会，以解决这一模型。我们开发了一种新的凝胶捕获策略来分离几乎纯的含有来源的片段，并准备了来自CHO和人类细胞的纯源文库。该提案的具体目的是：1)测试活性起源将被限制在转录活性的染色体区域，但将被限制在基因间区的提议。一个全面的人类起源文库(或未克隆的起始材料)将被用来在饱和条件下探测来自人类CHR 21和22的高密度微阵列。活性起始点相对于活性基因的分布将通过探测来自相同细胞的cDNA的微阵列来确定。这些研究还将提供固定地点与区域的活动起源的初步分类。2)制备高分辨率起始点准备，以确定与复制子相对应的固定起始点。凝胶捕获程序将被改进，以允许分离更小的含有起源的片段；或者，非常短的、以起源为中心的新生DNA将在体外合成在S早期的核中。所产生的材料将被用作CRR 21和22阵列上的探测器。固定起源相对于活性基因的分布将表明它们是在发育调节基因或基因簇的邻域中特定进化的。3)分离早期、中期和晚期的起源，并确定它们的激活时间与它们的遗传和表观遗传特征的关系。将在选定的时间点通过标准的气泡捕获程序从同步的细胞中制备起源文库，并将其与CHR 21和22阵列杂交。与AIMS 1和2的结果进行比较，将表明局部转录对起源激活时间的不同影响，以及起源利用的明显效率。4)使用计算方法识别固定起源中最常见的序列基序，并确定它们的位置在人、小鼠和大鼠中是否保守。将分析那些在AIMS 2和3中描述的似乎对应于单个位点或限定区域的起源，以寻找共同的组成偏向、周期性、折叠潜力、DNA解离元件和传统的序列基序，以揭示可能起复制功能的共性。它们在人类、小鼠和大鼠之间的保守性也将通过标准计算方法在基因组数据库中的比较来确定。5)验证活性起源和基因位于具有独特结构的共同染色质结构域的假设。修饰的组蛋白和选定的其他蛋白质在CHR 21和22上的分布将通过ChlP-on-Chip方法进行分析，使用各种相关蛋白质的抗体。通过将这些数据与目标1中确定的分布进行比较，我们将定义染色质结构的各个方面，这些方面表征了活性起源、起源簇和/或局部基因。

DESCRIPTION (provided by applicant): There are >50,000 active origins in the typical somatic genome. Owing to this complexity, only a few origins have been identified by molecular biological approaches, and then only after enormous cost and expenditure of effort. Many genes are replicated early in S-phase in cell types in which they are active but late when they are inactive, suggesting interplay between origin activity and transcription during development. Based on biochemical analyses of the handful of validated origins, there appear to be two types: 1) broad zones of inefficient initiation sites, and 2) highly preferred start sites. We propose that the genome is peppered at intervals of 1 kb or less with a hierarchy of potential initiation sites, a subset of which has evolved into true replicators. Usage of any given site is proposed to be regulated by local gene activity and chromatin architecture. The completion of high-quality human, murine, and rat genome sequences, as well as the advent of microarrays, provides a unique opportunity to perform global analyses of the distribution, structure, and regulation of replication origins in order to address this model. We have developed a novel gel-trapping strategy for isolating virtually pure origin-containing fragments, and have prepared pure libraries of origins from CHO and human cells. Specific aims of the proposal are: 1) To test the proposal that active origins will be confined to transcriptionally-active chromosomal domains, but will be confined to intergenic regions. A comprehensive human origin library (or the uncloned starting material) will be used to probe high-density microarrays from human chr 21 and 22 under saturating conditions. The distribution of active origins vis-a-vis active genes will be determined by probing the microarrays with cDNAs from the same cells. These studies will also provide a preliminary assortment of active origins into fixed sites versus zones. 2) To prepare high-resolution origin preparations to identify fixed initiation sites that could correspond to replicators. The gel-trapping procedure will be refined to allow isolation of smaller origin-containing fragments; alternatively, very short, origin-centered, nascent DNAs will be synthesized in vitro in early-S-phase nuclei. The resulting materials will be used as probes on the chr 21&22 arrays. The distribution of fixed origins vis-a-vis active genes will indicate whether they have evolved specifically in the neighborhoods of developmentally regulated genes or gene clusters. 3) To isolate early-, mid-, and late-firing origins and determine how their activation times relate to their genetic and epigenetic signatures. Origin libraries will be prepared from synchronized cells at selected time points by the standard bubble-trapping procedure and hybridized to the chr 21&22 arrays. Comparison to the results of Aims 1&2 will indicate differential effects of local transcription on the time of origin activation, as well as the apparent efficiency of origin utilization. 4) To use computational approaches to identify the most common sequence motifs among fixed origins, and determine whether their positions are conserved among humans, mice, and rats. Those origins characterized in Aims 2&3 that appear to correspond to single sites or circumscribed zones will be analyzed for common compositional bias, periodicity, fold-back potential, DNA unwinding elements, and conventional sequence motifs to uncover commonalities that might serve a replicator function. Their conservation among humans, mice, and rats will also be determined by comparisons among the genome databases by standard computational methodologies. 5) To test the hypothesis that active origins and genes reside in common chromatin domains with unique architectures. The distributions of modified histones and selected other proteins on chr 21 and 22 will be analyzed by the ChlP-on-ChIP approach, using a variety of antibodies to relevant proteins. By comparing these data to the distributions identified in Aim 1. we will define aspects of chromatin architecture that characterize active origins, origin clusters, and/or local genes.