Mechanisms of chromosome segregation, aneuploidy, and tumorigenesis

项目总结 在细胞分裂过程中，染色体是遗传的基本单位，传递到每个子细胞 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因素是由表观遗传而不是由DNA决定的 序列。在过去的10年里，我们已经确定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突变体CENP-A组装，鉴定其负载伴侣 HJURP，并确定着丝粒染色质只在有丝分裂结束时复制，在细胞周期结束后半个月 着丝粒DNA复制。在未来五年，将采取多个方向来确定如何 着丝粒的特性在表观遗传学上被复制和保持，包括全基因组分析以确定 分子事件调节我们已经确定的纠错机制，从而维持 着丝粒染色质与CENP-A组装，但将CENP-A错误加载到非着丝粒位点。 染色体错误分离或胞质分裂中的错误会产生非整倍体，这是一种不同于 单倍体数量的倍数。一项主要的努力将集中在确定正常背后的机制 染色体分离和在正常情况下防止非整倍体的行为，并检测 单个染色体错误分离或纺锤体极扩增在推动肿瘤发生中的后果。 我们以前已经鉴定了着丝粒特异的微管依赖的马达CENP-E，并确定它是 是一种真正的微管尖端追踪运动蛋白，并证明了它的限量产生广泛的， 细胞和小鼠的全染色体非整倍体。我们已经用所有提纯的组件进行了重建 以及在细胞和小鼠中进行基因靶向/沉默以确定支持有丝分裂的关键分子机制 检查点(也称为纺锤体组件检查点)，主要防御染色体 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退化标记，以确定着丝粒复制、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 ATPase的联合作用 TRIP13在有丝分裂检查点抑制物(S)的催化解体和/或有丝分裂检查点的初始激活中起作用。 非整倍体与肿瘤发生的联系早已被认识到，非整倍体在人类中也很常见。 癌症。伟大的德国细胞学家西奥多·博弗里最初提出了相关假说，即非整倍体 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导致肿瘤的发生 中心体扩增。使用从降低水平中高频错误分离染色体的小鼠 对于着丝粒运动蛋白CENP-E，我们以前证明了整个染色体的非整倍体可以 在某些遗传背景下促进肿瘤发生，但不影响由基因突变引起的肿瘤发生 DNA修复，当与增加非整倍体的遗传损伤结合时，延迟肿瘤的发生。我们 现在将测试中心体扩增如何影响肿瘤发生。使用我们已有的条件鼠标模型 产生额外的中心体可以瞬时诱导，我们将确定中心体是否 扩增促进细胞转化或自发性肿瘤的形成，能够促进 致癌物诱发的肿瘤的发展，并能够加速发展(或增加 肿瘤的侵袭性或转移潜能)是由肿瘤抑制基因的丢失引起的。 与染色体错误分离相关的一种染色体异常是染色质病(也称为 染色体再发生)，一条(或两条)染色体看起来已经分裂成十条到 数百个小的基因组片段，并以随机的顺序重新组合在一起。致变色 染色体是通过测序确定的，现在被认为存在于广泛的 癌症。对人类细胞和遗传植物模型的研究表明，最初的错误分离是 微核可引发嗜铬细胞症。我们现在建议使用一种新的方法来测试染色体病的机制 一种高效地产生特定染色体(Y)错分离为微核的方法。通过 利用人类Y着丝粒的一个独特特征，我们已经制造出了可以在其中产生 Y着丝粒选择性瞬间失活，Y染色体错误分离成微核 频率很高。我们将使用这种方法来确定持续性和/或暂时性着丝粒 灭活可从微核内碎裂的染色体中稳定地产生可遗传的染色细胞症 为了确定碎片化微核染色体重组的修复机制 会产生嗜铬细胞症。与此相关的是，新的方向将是识别染色体破碎和 在获得性耐药期间导致基因扩增的重组事件，包括产生 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.