Control of genetic and epigenetic instabilities by lincRNA genes

 描述（由申请人提供）：癌细胞在许多重要特征上不同于其正常细胞对应物。癌症的这些“标志”影响许多生物过程，并在人类癌症的多步发展过程中获得。毫不奇怪，遗传和/或表观遗传变化存在于大多数（如果不是所有）癌症中，并且已知是这些表型改变的原因。此外，遗传和/或表观遗传不稳定性被认为存在于大多数癌症中，并且被认为是产生个体肿瘤细胞中存在的许多遗传和/或表观遗传变化所需的。理解驱动这些改变的机制显然是一个重要的目标。我的实验室以前描述了与某些肿瘤衍生的染色体重排相关的染色体异常。这种异常表型影响单个染色体，其特征在于延迟复制时间（DRT），延迟有丝分裂染色体凝聚（DMC）和受影响染色体的频繁重排。我们开发了一种“染色体工程”系统，使我们能够以有效和可重复的方式生成DRT/DMC染色体。因此，使用基于Cre/loxP的策略来筛选人类染色体中的重排，我们鉴定了5个平衡易位，影响8个不同的常染色体，均显示DRT/DMC。我们还发现，工程DRT/DMC染色体显示染色体结构不稳定。最近，我们发现Cre/loxP介导的长非编码RNA基因（我们将其命名为ASAR 6）的破坏导致人类6号染色体上的DRT/DMC。我们还发现ASAR 6与Xist非编码RNA基因具有许多共同特征，包括：随机单等位基因表达，异步复制，顺式其他单等位基因沉默，以及在基因组转基因异位整合后延迟整个染色体复制的能力。值得注意的是，Xist的缺失导致延迟复制、异常染色质结构和X染色体上的二级重排。因此，ASAR 6的破坏导致Xist缺失的表型。此外，最近显示Xist的破坏在小鼠中引起100%渗透性造血恶性肿瘤，表明Xist作为肿瘤抑制剂起作用。最近，我们发现了第二个非编码RNA基因，暂时命名为ASAR 15，它控制着人类15号染色体的复制时间和稳定性。总之，这些观察结果提出了一种有趣的可能性，即所有哺乳动物染色体都含有“Xist样”肿瘤抑制基因座，其功能是维持个体染色体的遗传和表观遗传稳定性。构成该提议的实验被设计为：1）表征与ASAR 6、ASAR 15或Xist破坏相关的结构重排的“分子特征”; 2）阐明与ASAR 6或ASAR 15破坏相关的染色质和基因表达变化;以及3）鉴定具有ASAR 6、ASAR 15或Xist中的结构重排的人癌细胞。

 DESCRIPTION (provided by applicant): Cancer cells differ from their normal cellular counterparts in many important characteristics. These `hallmarks' of cancer affect numerous biological processes, and are acquired during the multistep development of human cancer. Not surprisingly, genetic and/or epigenetic changes are present in most if not all cancers, and are known to be responsible for these phenotypic alterations. In addition, genetic and/or epigenetic instabilities are thought to be present in most cancers and are thought to be required to generate the numerous genetic and/or epigenetic changes that are present in individual tumor cells. Understanding the mechanisms that drive these alterations is clearly an important goal. My lab previously characterized a chromosomal abnormality associated with certain tumor-derived chromosome rearrangements. This abnormal phenotype affects individual chromosomes and is characterized by delayed replication timing (DRT), delayed mitotic chromosome condensation (DMC), and frequent rearrangement of the affected chromosomes. We have developed a `chromosome engineering' system that allows us to generate DRT/DMC chromosomes in an efficient and reproducible manner. Thus, using a Cre/loxP-based strategy to screen for rearrangements in human chromosomes we identified 5 balanced translocations, affecting 8 different autosomes, all displaying DRT/DMC. We also found that the engineered DRT/DMC chromosomes display chromosome structure instability. More recently, we found that Cre/loxP-mediated disruption of a long non-coding RNA gene, which we named ASynchronous replication and Autosomal RNA on chromosome 6 (ASAR6), results in DRT/DMC on human chromosome 6. We also found that ASAR6 shares many characteristics with the Xist non-coding RNA gene, including: random monoallelic expression, asynchronous replication, silencing of other monoallelic genes in cis, and the ability to delay replication of entire chromosomes following ectopic integration of genomic transgenes. Notably, deletion of Xist results in delayed replication, abnormal chromatin structure and secondary rearrangements on the X chromosome. Therefore, disruption of ASAR6 results in a phenocopy of Xist deletion. Furthermore, disruption of Xist was recently shown to cause a 100% penetrant hematopoetic malignancy in mice, indicating that Xist functions as a tumor suppressor. More recently, we identified a second non-coding RNA gene, tentatively named ASAR15, which controls replication timing and stability of human chromosome 15. Taken together, these observations raise the intriguing possibility that all mammalian chromosomes contain `Xist-like' tumor suppressor loci functioning to maintain the genetic and epigenetic stability of individual chromosomes. The experiments that make up this proposal are designed to: 1) characterize the "molecular signature" of structural rearrangements associated with ASAR6, ASAR15 or Xist disruption; 2) elucidate the chromatin and gene expression changes associated with ASAR6 or ASAR15 disruption; and 3) identify human cancer cells with structural rearrangements in ASAR6, ASAR15 or XIST.