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.

项目概要 在细胞分裂过程中，染色体（遗传的基本单位）传递到每个子细胞是由介导的 由着丝粒。与 DNA 序列至关重要的典型基因不同，在后生动物中，这个中心 确保染色体遗传的遗传元件是由表观遗传决定的，而不是由 DNA 决定的 顺序。在过去的 10 年里，我们已经确定了着丝粒身份的表观遗传标记是 染色质与着丝粒选择性组蛋白变体 CENP-A 组装，确定了其负载伴侣 HJURP，并确定着丝粒染色质仅在有丝分裂退出时复制，即半个细胞周期后 着丝粒 DNA 复制。未来五年，将采取多个方向来确定如何 着丝粒身份通过表观遗传学进行复制和维持，包括全基因组分析以识别 介导纠错机制的分子事件我们已经确定了哪些行为可以维持 着丝粒染色质与 CENP-A 组装，但将错误加载到非着丝粒位点的 CENP-A 剥离。 染色体错误分离或胞质分裂中的错误会产生非整倍性，即染色体内容不同于 单倍体数的倍数。主要努力将集中于确定正常现象背后的机制 染色体分离，并在正常情况下防止非整倍体并测试 单染色体错误分离或纺锤体极扩增在驱动肿瘤发生中的后果。 我们之前已经鉴定了着丝粒特异性微管依赖性运动CENP-E，确定它 是一种真正的微管尖端追踪驱动蛋白，并证明其数量有限会产生广泛的、 细胞和小鼠中的整个染色体非整倍性。我们使用了所有纯化成分的重建 以及细胞和小鼠中的基因靶向/沉默，以确定有丝分裂背后的关键分子机制 检查点（也称为纺锤体装配检查点），针对染色体的主要防御 哺乳动物中的错误分离。在接下来的5年里，我们建议使用CRISPR进行基因替换- Cas9 基因组编辑和生长素诱导的降解决定子标签可识别着丝粒复制、有丝分裂的关键方面 检查点激活和沉默功能，包括最初关注 AAA+ ATP 酶的联合作用 TRIP13 在有丝分裂检查点抑制剂的催化分解和/或初始有丝分裂检查点激活中的作用。 非整倍性与肿瘤发生的联系早已被认识到，非整倍性在人类中很常见 癌症。德国伟大的细胞学家Theodor Boveri最初提出非整倍体的相关假说 由于单个染色体的错误分离或由以下原因引起的异常有丝分裂而驱动肿瘤发生 中心体扩增。使用在降低水平的情况下以高频率错误分离染色体的小鼠 着丝粒运动蛋白CENP-E，我们之前表明，整个染色体非整倍体可以 在某些遗传背景下促进肿瘤发生，但不影响由突变引起的肿瘤发生 DNA 修复，并在与也会增加非整倍性的遗传损伤相结合时延迟肿瘤发生。我们 现在将测试中心体扩增如何影响肿瘤发生。使用条件小鼠模型，我们有 产生的额外中心体可以被短暂诱导，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 致癌物诱发的肿瘤的发展，并且能够加速发展（或增加 肿瘤抑制基因缺失导致的肿瘤的侵袭性或转移潜力。 与染色体错误分离有关的一种相关染色体异常是染色体碎裂（也称为 染色体发生），一个（或两个）染色体似乎被破碎成数十个的事件 数百个小的基因组片段并以随机顺序重新连接在一起。显色 染色体是通过测序鉴定出来的，现在被认为存在于广泛的染色体中。 癌症。对人类细胞和遗传植物模型的研究表明，最初的错误分离 微核可以引发染色体碎裂。我们现在建议使用 高效地将特定染色体（Y）错误分离成微核的方法。经过 利用人类 Y 着丝粒的独特特征，我们已经生产出可以生产 Y 着丝粒选择性、短暂失活，Y 染色体错误分离成微核 高频。我们将使用这种方法来确定是否持续和/或短暂着丝粒 失活可以从微核内碎片化的染色体中产生稳定的可遗传的染色体碎裂，并且 确定碎片微核染色体重新组装的修复机制 产生染色体碎裂。与此相关，新的方向将是识别染色体破碎和 获得性耐药期间基因扩增的重组事件，包括产生 双分钟或同质染色区域。