Mechanisms of chromosome segregation, aneuploidy, and tumorigenesis

染色体分离、非整倍性和肿瘤发生的机制

• 批准号: 9883009
• 负责人: Don W Cleveland
• 金额: $ 85.89万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883009
• 关键词: ATP phosphohydrolase; Affect; Aneuploidy; Automobile Driving; Auxins; Back; CENP-E protein; CRISPR/Cas technology; Carcinogens; Cell Cycle; Cell division; Cells; Centromere; Centrosome; Chromatin; Chromosome Segregation; Chromosome abnormality; Chromosomes; Cytokinesis; DNA Repair; DNA Sequence; DNA biosynthesis; Development; Double Minutes; Epigenetic Process; Event; Frequencies; Gene Amplification; Gene Targeting; Generations; Genes; Genetic; Genomics; German population; Haploidy; Heritability; Histones; Human; Individual; Joints; Kinesin; Lesion; Link; Malignant Neoplasms; Mammals; Mediating; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic Checkpoint; Mitotic spindle; Molecular; Molecular Chaperones; Motor; Mus; Mutation; Plant Model; Site; Stains; Testing; Tumor Suppressor Genes; Variant; Y Chromosome; acquired drug resistance; centromere protein A; chromosome missegregation; chromosome number abnormality; chromothripsis; daughter cell; gene replacement; genetic element; genome editing; genome-wide analysis; metaplastic cell transformation; mitotic checkpoint inhibitors; mouse model; plant genetics; prevent; reconstruction; repaired; tumor; tumorigenesis

PROJECT SUMMARY Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes for which the DNA sequence is crucial, in metazoans this central genetic element for insuring chromosome inheritance is determined epigenetically rather than by DNA sequence. Over the last 10 years, we have identified the epigenetic mark of centromere identity to be chromatin assembled with the centromere-selective histone variant CENP-A, identified its loading chaperone HJURP, and determined that centromeric chromatin is replicated only at exit from mitosis, half a cell cycle after centromere DNA replication. In the next five years, multiple directions will be undertaken for identifying how centromere identity is replicated and maintained epigenetically, including genome wide analyses to identify the molecular events that mediate an error correction mechanism we have identified which acts to maintain centromeric chromatin assembled with CENP-A, but strips CENP-A misloaded onto non-centromeric sites. Chromosome missegregation or errors in cytokinesis produce aneuploidy, a chromosome content other that a multiple of the haploid number. A major effort will focus on identifying the mechanisms underlying normal chromosome segregation and that act to prevent aneuploidy in the normal situation and testing the consequences of single chromosome missegregation or spindle pole amplification in driving tumorigenesis. We have previously identified the centromere-specific microtubule-dependent motor CENP-E, determined it to be a true microtubule tip tracking kinesin, and demonstrated that limiting amounts of it produce widespread, whole chromosomal aneuploidy in cells and in mice. We have used reconstruction with all purified components and gene targeting/silencing in cells and mice to identify key molecular mechanisms underlying the mitotic checkpoint (also known as the spindle assembly checkpoint), the primary guard against chromosome missegregation in mammals. In the upcoming 5 years, we propose to use gene replacement with CRISPR- Cas9 genome editing and auxin-inducible degron tags to identify key aspects of centromere replication, mitotic checkpoint activation and silencing function, including an initial focus on the joint action of the AAA+ ATPase TRIP13 in catalytic disassembly of mitotic checkpoint inhibitor(s) and/or initial mitotic checkpoint activation. The linkage of aneuploidy to tumorigenesis has long been recognized and aneuploidy is frequent in human cancers. The great German cytologist Theodor Boveri initially proposed related hypotheses that aneuploidy drives tumorigenesis from missegregation of individual chromosomes or an aberrant mitosis caused by centrosome amplification. Using mice that missegregate chromosomes at high frequency from reduced levels of the centromere motor protein CENP-E, we showed previously that whole chromosomal aneuploidy can facilitate tumorigenesis in some genetic contexts, but does not affect tumorigenesis caused by mutations in DNA repair, and delays tumorigenesis when combined with genetic lesions that also increase aneuploidy. We now will test how centrosome amplification affects tumorigenesis. Using a conditional mouse model we have produced in which extra centrosomes can be transiently induced, we will determine whether centrosome amplification promotes cellular transformation or the formation of spontaneous tumors, is capable of facilitating the development of carcinogen-induced tumors, and is able to accelerate the development (or increase the aggressiveness or metastatic potential) of tumors driven by the loss of a tumor suppressor gene. A related chromosomal abnormality linked to chromosome missegregation is chromothripsis (also known as chromoanagenesis), an event in which one (or two) chromosomes appear to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. Chromotriptic chromosomes were identified by sequencing and are now recognized to be present in a broad range of cancers. Efforts with human cells and genetic plant models have suggested that initial missegregation into micronuclei can trigger chromothripsis. We propose now to test mechanisms of chromothripsis using an approach to generate missegregation of a specific chromosome (the Y) into micronuclei at high efficiency. By exploiting a unique feature of the human Y centromere, we have produced cells in which we can produce selective, transient inactivation of the Y centromere, with the Y chromosome missegregated into micronuclei at high frequency. We will use this approach to determine whether sustained and/or transient centromere inactivation can produce stably heritable chromothripsis from chromosomes fragmented within micronuclei and to determine the repair mechanisms underlying reassembly of fragmented micronuclear chromosomes to generate chromothripsis. Related to this, new directions will be to identify the chromosome shattering and reassembly events that underlie gene amplification during acquired drug resistance, including generation of double minutes or homogenous staining regions.

项目摘要 在细胞分裂期间，染色体的输送（遗传的基本单位）是介导的 由Centromere。与典型的基因DNA序列至关重要，在后代中 确保染色体遗传的遗传元素是表观遗传学的，而不是通过DNA确定的 顺序。在过去的十年中，我们确定了Centromere身份的表观遗传标记为 染色质与中心粒选择性组蛋白变体CENP-A组装，鉴定了其负载伴侣 Hjurp，并确定仅在有丝分裂的出口时复制了丝粒染色质，一半的细胞周期 Centromere DNA复制。在接下来的五年中，将为确定如何确定多个方向 在表观遗传上复制并维持着丝粒的身份，包括基因组广泛的分析，以鉴定 介导误差校正机制的分子事件，我们已经确定了要维护的作用 丝粒染色质与CENP-A组装在一起，但带有CENP-A的cenP-A误后载于非中心位点。 染色体的错误分析或细胞因子的错误会产生非整倍性，染色体含量是其他 单倍体数的倍数。一项重大努力将集中于确定正常机制 染色体隔离和该行为在正常情况下预防非整倍性并测试 在驱动肿瘤发生中，单个染色体错误分析或主轴放大的后果。 我们先前已经确定了丝粒特异性微管依赖性运动CENP-E，确定了它 成为真正的微管尖端跟踪运动蛋白，并证明其限制量会产生广泛的量 细胞和小鼠中的整个染色体非整倍性。我们已经使用所有纯化组件的重建 细胞和小鼠中的基因靶向/沉默，以鉴定有丝分裂的关键分子机制 检查点（也称为主轴装配检查站），反对染色体的主要防护 哺乳动物的错误分析。在接下来的5年中，我们建议将基因替代者与CRISPR一起使用 CAS9基因组编辑和生长素诱导的DEGRON标签，以识别丝粒复制的关键方面 检查点激活和沉默函数，包括对AAA+ ATPase联合作用的初始重点 TRIP13在有丝分裂检查点抑制剂和/或初始有丝分裂检查点激活的催化拆卸中。 长期以来已经识别出非整倍性与肿瘤发生的联系，而非整倍性在人类中经常存在 癌症。德国伟大的细胞学家西奥多·博维里（Theodor Boveri）最初提出的相关假设是非整倍性的 从单个染色体的错误分析或由异常有丝分裂引起的肿瘤发生 中心体扩增。使用从降低的高频以高频进行染色体的小鼠 在Centromere运动蛋白CENP-E中，我们以前表明整个染色体非整倍性可以 在某些遗传环境中促进肿瘤发生，但不影响由突变引起的肿瘤发生 DNA修复，并延迟肿瘤发生，并结合遗传病变也会增加舌骨。我们 现在将测试中心体扩增如何影响肿瘤发生。使用条件鼠标模型我们有 产生的，可以暂时诱导额外的中心体，我们将确定中心体是否 扩增促进细胞转化或自发肿瘤的形成，能够促进 致癌诱导的肿瘤的发展，并能够加速发育（或增加 受肿瘤抑制基因丧失驱动的肿瘤的侵略性或转移潜力。 与染色体错误分析有关的相关染色体异常是染色体（也称为 染色体），一个事件，其中一个（或两个）染色体似乎已被粉碎成十亿 数百个小基因组碎片和宗教以随机的顺序恢复在一起。圆旋性 通过测序鉴定染色体，现在被认为存在于广泛范围内 癌症。人类细胞和遗传植物模型的努力表明，最初的错误分析到 微核可以触发染色体。我们现在建议使用一个 以高效率将特定染色体（Y）的错误进行分解的方法。经过 利用人类Y Centromere的独特特征，我们产生了可以产生的细胞 Y centromere的选择性，瞬时灭活，Y染色体错误地归因于Microduclei 高频。我们将使用这种方法来确定是否持续和/或瞬态中心 失活会产生稳定的可遗传性铬骨，从微核中碎片的染色体和 确定零散的微核染色体重新组装的修复机制 产生Chromothripsis。与此相关的是，新的方向将是确定染色体破碎和 重新组装事件是基因在获得耐药性期间基因扩增的基础的，包括生成 双分钟或同质染色区域。