Human Artificial Chromosomes for Cancer Research and Functional Genomics

由α DNA阵列组装的人类人工染色体（humanartificialchromosomes，HACs）是一种新型载体，在人类着丝粒的组装和维持研究、基因治疗、抗癌药物筛选和生物技术等方面具有巨大的应用潜力。我们先前构建了一个合成的HAC（tetO-HAC），允许通过不同的染色质修饰与tet-阻遏蛋白融合来束缚其动粒。这种HAC被成功地用于阐明不同类型的染色质在动粒功能中的作用。在过去的一年中，基于通过不同融合系留tetO-HAC动粒的相同方法揭示了有丝分裂转录加上组蛋白修饰（包括H3 K9 ac）构成了允许CENP-A组装和中心染色质维持的“表观遗传景观”。H3 K4 me 2是转录所必需的，H3 K9 ac可以形成屏障，以防止异染色质扩散和着丝粒失活。在另一项研究中，我们证明了人着丝粒抵抗H3 K27 me 3/K9 me 3介导的沉默。在单独的实验中，具有多整合酶重组位点的平台已被插入tetO-HAC中，并已成功用于HAC中的基因组装。工作正在进行中，以组装合成核仁组织区（NOR）的HAC使用TAR分离的人rDNA单位。这种HAC模块可通过微细胞介导的染色体转移（MMCT）转移到任何类型的人细胞中，将用于研究rDNA重复序列的核仁位置的要求以及rDNA单元的拷贝数对细胞增殖和应激反应的影响。我们还将我们的tetO-HAC用于测量人类细胞中的染色体不稳定性（CIN）。全染色体不稳定性（CIN），表现为细胞分裂期间染色体分布不均匀，是大多数类型癌症的特征，因此将其与正常对应物区分开来。尽管CIN通常被认为是肿瘤生长的驱动因素，但存在阈值水平，由此CIN频率的进一步增加成为肿瘤生长的障碍，因此可以在治疗上加以利用。然而，已知增加CIN超过该治疗阈值的药物目前数量很少。在我们以前的工作中，我们已经开发了一种新的定量测定CIN的基础上使用的非必需的HAC携带组成型表达的EGFP转基因。因此，继承HAC的细胞显示绿色荧光，而缺乏HAC的细胞不显示。这允许通过常规流式细胞术测量响应于药物治疗的HAC损失率。我们使用该试验对100多种抗癌药物对HAC损失的影响进行了排名。观察到紫杉醇（微管稳定剂）、LMP 744（拓扑异构酶TOP 1抑制剂，在我们的分支中开发）的作用最强。我们还证明了该测定在控制染色体传递的已知基因的siRNA耗尽后检测CIN增加的实用性。在我们最近的工作中，我们修改了EGFP-HAC，并将原来的检测转化为化学文库和人基因siRNA文库的高通量CIN筛选。针对720个人类蛋白激酶基因中的每一个的siRNA分析揭示了27个CIN基因，没有关于它们在染色体传递中的作用的信息。这些新的CIN基因中的每一个都可以被认为是癌症治疗的新靶点。

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study assembly and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its kinetochore by different chromatin modifies fused with the tet-repressor protein. This HAC was successfully used to clarify role of different types of chromatin in functioning of the kinetochore. During past year, the same approach based on tethering of the tetO-HAC kinetochore by different fusions revealed that mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres. In another study, we demonstrated that human centromere resists silencing mediated by H3K27me3/K9me3. In separate experiments, a platform with multi-integrase recombination sites has been inserted into tetO-HAC and has been successfully used for a gene assembly in the HAC. Work is in progress to assemble synthetic nucleolar organizer region (NOR) in the HAC using TAR-isolated human rDNA units. Such a HAC module that is transferable into any type of human cells via micro-cell mediated chromosome transfer (MMCT) will be used to investigate the requirements for nucleolar location of rDNA repeats and effect of copy number of rDNA units on cell proliferation and stress response. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a non-essential HAC carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows measurement of HAC loss rate in response to drug treatment by routine flow cytometry. We used this assay to rank more than 100 anticancer drugs on their effect on HAC loss. The strongest effect was observed for, taxol (microtubule-stabilizing agent), LMP744 (inhibitor of topoisomerase TOP1, developed in our branch). We also demonstrated the utility of the assay to detect increase of CIN after siRNA depletion of known genes controlling chromosome transmission. In our recent work, we modified EGFP-HAC and converted the original assay into high-throughput CIN screen of chemical libraries and siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed 27 CIN genes with no information on their role in chromosome transmission. Each of these new CIN genes may be considered as a new target for cancer therapy.