Variation, functions, and dynamics of human subtelomeres

描述（由申请人提供）：亚端粒可能是人类基因组中结构最复杂、最可变和最动态的区域。亚端粒交换将序列分布到多个染色体末端，并导致亚端粒含量的广泛变化。信息仍然是不完整的，但亚端粒含有基因，使个体间变异成为表型多样性的潜在来源。亚端粒同源性也可能介导有害的重排导致疾病和/或在ALT途径中发挥作用，使癌细胞在没有端粒酶的情况下无限增殖。我们的长期目标是了解亚端粒的不寻常特征如何影响表型多样性、物种形成和疾病。我们的三个目标是表征(1)结构，变异性和进化，(2)基因内容和功能，以及(3)亚端粒的有丝分裂和减数分裂动力学。该项目建立在我们在FISH、基因组分析和染色体分选方面的专业知识的基础上，并补充了其他地方正在进行的对每个亚端粒的代表性等位基因进行测序的努力。目标1有四个子目标。（1a）通过使用计算和实验相结合的方法，我们将确定多拷贝块的边界，并评估其数量和位置的多态性。（1b）我们将测试亚端粒复制仅在最近才传播的假设，通过使用FISH来定位其他灵长类动物和新旧世界猴子的各种块。（1c）我们将从几个物种的“同源”亚端粒区分离并绘制克隆图谱，由埃里克·格林（Eric Green）的团队进行测序，以确定在人类与其他灵长类动物分化时可能丢失的序列。（1d）并且，作为原理证明，我们将对主要存在于非洲人身上的变异7p等位基因进行有针对性的克隆和测序。在目标2中，我们将分析约20个亚端粒基因家族的潜在功能和表达，并评估人类和灵长类动物之间基因序列和数量的变化。在目标3中，我们将分析亚端粒区域在重组事件中的作用。（3a）我们将进行CO-FISH分析，以确定亚端粒区域内的姐妹染色单体交换（SCE）是否导致了染色体末端带异常高的SCE频率。（3b）我们将开发和部署一种检测正常细胞、DNA修复缺陷细胞和ALT细胞亚端粒间染色体间有丝分裂交换的方法。并且，我们将确定染色体末端的减数分裂错配与亚端粒序列同源性的关系。我们对人类基因组这些复杂区域的基因组和功能分析应该为亚端粒动力学的双刃剑在调节适应性变化和有害重排方面的尖锐程度提供新的见解。

DESCRIPTION (provided by applicant): Subtelomeres are likely the most structurally complex, variable, and dynamic regions of the human genome. Subtelomeric exchanges have distributed sequences to multiple chromosome ends and resulted in extensive variation in subtelomeric content. Information is still fragmentary, but subtelomeres contain genes, making interindividual variation a potential source of phenotypic diversity. Subtelomeric homology may also mediate deleterious rearrangements to cause disease and/or play a role in the ALT pathway, enabling cancer cells to proliferate indefinitely without telomerase. Our long-term goal is to understand how the unusual characteristics of subtelomeres impact phenotypic diversity, speciation, and disease. Our three aims are to characterize the (1) structure, variability, and evolution, (2) gene content and function, and (3) mitotic and meiotic dynamics of subtelomeres. The project builds on our expertise in FISH, genome analysis, and chromosome sorting and complements efforts being made elsewhere to sequence a representative allele of each subtelomere. Aim 1 has four sub-aims. (1a) By using a combined computational and experimental approach, we will determine the boundaries of multicopy blocks and assess polymorphism in their number and location. (1b) We will test the hypothesis that subtelomeric duplications spread only recently, by using FISH to locate various blocks in other primates and Old and New World monkeys. (1c) We will isolate and map clones from "orthologous" subtelomeric regions in several species for sequencing by Eric Green's group, in order to identify sequences that may have been lost as humans diverged from other primates. (1d) And, as a proof of principle, we will perform targeted cloning and sequencing of a variant 7p allele found predominantly in Africans. In Aim 2, we will analyze each of ~20 subtelomeric gene families for potential function and expression and assess variation in gene sequence and number among humans and primates. In Aim 3, we will analyze the involvement of subtelomeric regions in recombination events. (3a) We will conduct CO-FISH assays to determine if sister chromatid exchanges (SCEs) within subtelomeric zones account for the unusually high SCE frequencies attributed to terminal bands of chromosomes. (3b) We will develop and deploy an assay for inter-chromosomal mitotic exchange between subtelomeres in normal, DNA repair-deficient, and ALT cells. (3c) And, we will determine how meiotic mispairing of chromosome ends relates to subtelomeric sequence homology. Our genomic and functional analyses of these complex regions of the human genome should provide new insights into how sharp the double-edged sword of subtelomere dynamics is in mediating adaptive change and deleterious rearrangements.