The role of ASARs in chromosome dynamics

摘要/摘要 哺乳动物细胞在不同的时间在每条染色体上的多个位置启动DNA复制， 一个时间复制程序。我们已经使用染色体工程策略来识别顺式作用基因座 它们控制着个体人类染色体上的复制计时程序。我们发现Cre/loxP- 影响八种不同常染色体的介导性易位，表现出复制时间和结构上的延迟 整个染色体的不稳定。随后，我们发现Cre/loxP介导的lncRNA的破坏 基因ASAR6和ASAR15分别导致人类6号和15号染色体的复制延迟。 ASAR6和ASAR15有许多共同的特征，包括：1)随机单等位基因表达 LncRNAs可以在物理上将整个染色体“包裹”在顺式基因中；2)等位基因之间的异步复制；3) 基因突变导致它们各自的染色体结构不稳定；以及4)异位整合 转基因导致顺式染色体中整个染色体的复制延迟。在早期的研究中，我们检测到 大量人类和人类基因重排后的延迟复制和结构不稳定性表型 小鼠染色体，这表明所有哺乳动物的染色体都受类似的机制调控。我们的 研究表明，所有哺乳动物的染色体都受相似的基因座调控。因此，我们提议 所有哺乳动物的染色体都含有“失活/稳定中心”(I/SCs)，它们的正常功能是 促进个体正确的复制时机、单等位基因表达和结构稳定性 染色体。我们认为，I/SCs对哺乳动物染色体生物学的根本重要性不亚于 端粒、着丝粒或复制起点。因此，在这种情况下，每个哺乳动物的染色体 包含四个基本的顺式作用元件，起源、着丝粒、端粒和I/SC，所有这些元件都起到 确保每条染色体的适当复制、分离和稳定。这项提议旨在 阐明ASAR6和ASAR15 RNA控制染色体复制时间的机制，以 确定ASAR6和ASAR15 RNA的染色质相互作用部位，并确定其作用机制 ASAR6和ASAR15的功能是在细胞核的3D空间内定位染色体，如果它们控制 6号和15号染色体的基因组相互作用图。如果成功，本文件中描述的实验 该提议将有助于建立哺乳动物染色体生物学的新范式，并将作为 为第四类基本染色体元件，即“失活/稳定中心”奠定了基础。

Summary/Abstract Mammalian cells initiate DNA replication at multiple sites along each chromosome at different times, following a temporal replication program. We have used a chromosome-engineering strategy to identify cis-acting loci that control this replication-timing program on individual human chromosomes. We found that Cre/loxP- mediated translocations, affecting eight different autosomes, display a delay in replication timing and structural instability of entire chromosomes. Subsequently, we found that Cre/loxP-mediated disruption of the lncRNA genes ASAR6 and ASAR15 result in delayed replication of human chromosomes 6 and 15, respectively. ASAR6 and ASAR15 share numerous characteristics, including: 1) random mono-allelic expression of lncRNAs that can physically “coat” entire chromosomes in cis; 2) asynchronous replication between alleles; 3) genetic disruption results in structural instability of their respective chromosomes; and 4) ectopic integration of transgenes causes delayed replication of entire chromosomes in cis. In earlier studies, we detected the delayed replication and structural instability phenotypes following rearrangement of numerous human and mouse chromosomes, suggesting that all mammalian chromosomes are regulated by similar mechanisms. Our work suggests that all mammalian chromosomes are regulated by similar loci. Therefore, we are proposing that all mammalian chromosomes contain “Inactivation/Stability Centers” (I/SCs), which normally function to promote proper replication timing, monoallelic gene expression and structural stability of individual chromosomes. We believe that I/SCs are as fundamentally important to mammalian chromosome biology as telomeres, centromeres, or origins of replication. Thus, under this scenario every mammalian chromosome contains four essential cis-acting elements, origins, centromeres, telomeres, and I/SCs all functioning to ensure proper replication, segregation and stability of each chromosome. This proposal is designed to elucidate the mechanisms by which ASAR6 and ASAR15 RNAs control chromosome replication timing, to identify chromatin interaction sites of ASAR6 and ASAR15 RNAs, and to determine the mechanisms by which ASAR6 and ASAR15 function to localize chromosomes within the 3D space of the nucleus, and if they control the genome interaction maps of chromosomes 6 and 15. If successful the experiments described in this proposal will help to establish a new paradigm for mammalian chromosome biology, and will serve as the foundation for a fourth type of essential chromosomal element, the “Inactivation/Stability Center”.