Mechanisms of chromosome segregation, aneuploidy, and tumorigenesis

项目摘要 在细胞分裂过程中，染色体（遗传的基本单位）被介导传递到每个子细胞 由着丝粒组成。与DNA序列至关重要的典型基因不同，在后生动物中， 确保染色体遗传的遗传因素是由表观遗传学而不是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，鉴定其装载伴侣蛋白 HJURP，并确定了着丝粒染色质仅在有丝分裂结束时复制，即在有丝分裂后的半个细胞周期。 着丝粒DNA复制。在今后五年中，将采取多个方向，以确定如何 着丝粒身份是复制和维持表观遗传，包括全基因组分析，以确定 我们已经确定了介导错误纠正机制的分子事件， 着丝粒染色质与CENP-A组装，但将CENP-A错载到非着丝粒位点上。 染色体错误分离或胞质分裂中的错误产生非整倍体，即染色体内容不同于 的倍数。一项主要的工作将集中在确定正常的潜在机制， 染色体分离，并在正常情况下防止非整倍性，并测试 单个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。 我们以前已经确定了着丝粒特异性微管依赖性运动CENP-E，确定它 是一个真正的微管尖端跟踪驱动蛋白，并证明，有限的数量，它产生广泛， 细胞和小鼠中的全染色体非整倍性。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，以鉴定有丝分裂的关键分子机制。 检查点（也称为纺锤体组装检查点），是防止染色体 哺乳动物的错误隔离在接下来的5年里，我们计划使用CRISPR进行基因替换- Cas9基因组编辑和生长素可诱导的降解决定子标签，以识别着丝粒复制、有丝分裂 检查点激活和沉默功能，包括对AAA+ ATP酶联合作用的初步关注 TRIP 13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。 非整倍体与肿瘤发生的关系早已被人们所认识，并且非整倍体在人类中是常见的 癌的伟大的德国细胞学家Theodor Boveri首先提出了相关假说， 由单个染色体的错误分离或异常有丝分裂引起的肿瘤发生， 中心体扩增使用小鼠，在高频率错误分离染色体从降低水平 着丝粒运动蛋白CENP-E，我们以前表明，整个染色体非整倍性， 在某些遗传背景下促进肿瘤发生，但不影响 DNA修复，并延迟肿瘤发生时，结合遗传病变，也增加非整倍体。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 产生额外的中心体可以瞬时诱导，我们将确定是否中心体 扩增促进细胞转化或自发性肿瘤的形成，能够促进 致癌物诱导的肿瘤的发展，并能够加速发展（或增加 侵袭性或转移潜力）。 与染色体错误分离相关的染色体异常是染色体碎裂（也称为染色体碎裂）。 染色体再生（chromoanagenesis），一个（或两个）染色体似乎已经破碎成数十个， 数百个小的基因组片段，并以随机顺序重新连接在一起。变色的 染色体是通过测序鉴定的，现在被认为存在于广泛的 癌的对人类细胞和遗传植物模型的研究表明，最初的错误分离到 微核可引发染色体断裂。我们现在建议使用一种新的方法来测试chromothripsis的机制。 一种以高效率将特定染色体（Y）错误分离成微核的方法。通过 利用人类Y染色体着丝粒的一个独特特征， Y着丝粒的选择性瞬时失活，Y染色体在 高频率我们将使用这种方法来确定是否持续和/或短暂的着丝粒 失活可从微核内断裂的染色体产生稳定遗传的染色体断裂， 以确定修复机制潜在的重组碎片微核染色体， 产生色裂。与此相关，新的方向将是确定染色体破碎， 在获得性耐药性期间，作为基因扩增基础的重组事件，包括 双分钟或均匀染色区域。

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.