Human Artificial Chromosomes for Cancer Research and Functional Genomics

人类人工染色体(HAC)是一种新型载体，在人类动粒的研究、组装和维持以及基因治疗、抗癌药物筛选和生物技术等方面具有巨大的潜力。我们以前构建了一个人工合成的HAC(Teto-HAC)，允许通过不同的染色质修饰物与tet抑制蛋白融合来拴系其动粒。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。在过去的一年里，同样的方法基于不同融合对Teto-HAC动粒的拴系，揭示了有丝分裂转录和包括H3K9ac在内的组蛋白修饰构成了允许CENP-A组装和中心染色质维持的“表观遗传格局”。H3K4me2是转录所必需的，H3K9ac可能形成一种屏障，以防止人类着丝粒上的异染色质扩散和动粒失活。在另一项研究中，我们证明了人类着丝粒抵抗H3K27me3/K9me3介导的沉默。在单独的实验中，一个带有多整合酶重组位点的平台被插入到Teto-HAC中，并成功地用于HAC中的基因组装。使用焦油分离的人类rDNA单位在HAC中组装合成核仁组织者区域(NOR)的工作正在进行中。这种可以通过微细胞介导的染色体转移(MMCT)转移到任何类型的人类细胞中的HAC模块将被用于研究rDNA重复序列的核仁位置要求以及rDNA单位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(CIN)，表现为细胞分裂过程中染色体分布不均匀，是大多数类型癌症的特征，因此区别于正常癌症。尽管CIN通常被认为是肿瘤生长的驱动力，但存在一个阈值水平，由此CIN频率的进一步增加成为阻止肿瘤生长的障碍，因此可以用于治疗。然而，已知的增加CIN超过这一治疗阈值的药物目前数量很少。在我们之前的工作中，我们开发了一种新的定量检测CIN的方法，该方法基于携带结构性表达的EGFP转基因的非必需HAC的使用。因此，继承了HAc的细胞会显示绿色荧光，而没有HAc的细胞则不会。这使得可以通过常规的流式细胞术来测量药物治疗后的HAc损失率。我们使用这种方法对100多种抗癌药物的HAc丢失效果进行了排名。其中，紫杉醇(微管稳定剂)、LMP744(我所研制的TOP1拓扑异构酶抑制剂)作用最强。我们还证明了该方法在检测控制染色体传递的已知基因的siRNA缺失后CIN增加的实用价值。在我们最近的工作中，我们对EGFP-HAC进行了改进，将原来的方法转化为高通量的CIN筛选人类基因的化学文库和siRNA文库。针对720个人类蛋白激酶基因的siRNA分析显示，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基因都可能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.