Human Artificial Chromosomes for Cancer Research and Functional Genomics

人类人工染色体（Human Artificial chromosome, HACs）是一种新的载体，在人类着丝点的研究和维持、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。众所周知，HACs是在将阿尔法DNA阵列转染到人体细胞后产生的。形成HAC的效率很低，需要分析大量的侵染物。在我们最近的合作工作中，我们首次系统地研究了阿尔法DNA阵列对高效HAC形成的结构要求。我们证明了CENP-B盒阳性和阴性合成α卫星重复序列的组合极大地改善了从头HAC的形成。之前我们构建了一个合成的HAC (tetO-HAC)，通过不同的染色质修饰与Tet抑制蛋白融合，可以将HAC着丝点系在一起。着丝粒含有tetO重复序列的tetO- hac已被广泛用于研究着丝粒内的蛋白质相互作用，并定义着丝粒染色质的表观遗传特征。在我们最近的研究中，我们开发了一种新的合成HAC，它包含两个具有不同靶向序列的阿尔法DNA阵列，tetO和lacO，即lacO/tetO-HAC。这种新的HAC可以用于详细的表观遗传工程研究，并研究着丝粒在双中心染色体中重新定位和失活的机制。与其他HAC载体相比，tetO-HAC具有优势，因为它可以通过染色质修饰剂（如tTS）与着丝粒tetO序列结合，使HAC着丝粒失活，从而很容易从细胞中消除。在单独的实验中，一个具有多整合酶重组位点的平台被插入到tetO-HAC中，用于组装HAC中的大型遗传位点。我们正在进行的工作是在HAC中组装一个人工核仁组织者区（NOR），以阐明NOR与核仁结合的分子机制。为此，利用本实验室开发的转化相关重组（TAR）克隆方法，从人类染色体21和22中分离出45 kb rDNA单元和侧翼近端连接（PJ）和远端连接（DJ）序列。值得注意的是，对rDNA单元的分析显示，45S RNA区域的序列变异数量出乎意料地高。18S和28S rRNA区域的一些变异可能影响核糖体RNA折叠或与核糖体蛋白的相互作用。从22号染色体中分离出完整的rDNA阵列以及两侧的PJ和DJ区域，为合成NOR区域的组装提供了平台。这可以详细研究人类细胞核仁形成的序列要求和机制，包括PJ和DJ在这一过程中的作用。为了描述人类细胞核仁形成的序列要求，我们将22号染色体上对应完整45 kb rDNA单元、DJ和PJ区域的不同NOR片段插入人类HT1080中tetO-HAC繁殖的基因装载位点。为了确定携带不同TAR结构的HACs在间期细胞中的核仁结合状态，我们应用了3D免疫- fish。用tetO-alphoid阵列的PNA探针观察HACs。用抗Nop52抗体观察核仁。在携带45 kb完整rDNA重复序列和58 kb DJ区域的HAC中观察到核仁结合。相反，携带53kb PJ片段的HAC的核仁结合体百分比与携带GFP转基因的对照HAC没有差异。总的来说，我们对携带22号染色体不同NOR区域的HACs的分析表明，单个rDNA重复和58 kb的DJ DNA片段足以驱动这些HACs的核仁结合。利用携带rDNA单元和DJ区不同片段的HAC载体，阐明NORs与核仁关联的分子机制的工作正在进行中。染色体数目异常是大多数实体瘤的一个特征，通常伴随着染色体不稳定性（CIN）的升高。整个染色体的获得或丢失导致基因拷贝数和表达水平的大规模变化。CIN基因的突变被认为是肿瘤发展的早期事件。目前，大约有400个控制适当染色体传递的人类基因已经用基因本体术语进行了注释，而在酵母中系统的CIN基因筛选已经发现了900多个基因。因此，可以推测，许多人类CIN基因仍未被识别。在我们之前的工作中，我们开发了一种高通量的检测方法，用于鉴定新的人类CIN基因，使用tetO-HAC表达去生长不稳定的EGFP。在目前的研究中，我们正在使用NCATS Ambion收集的19,000个sirna，覆盖整个人类基因组，用于筛选新的CIN基因。结果，鉴定出250个新的CIN候选基因。250个CIN候选基因的再确认实验正在进行中。第一步是使用新开发的sirna敲除基因。新的CIN基因的鉴定应该为开发新的治疗策略创造机会，以靶向癌细胞的CIN表型。端粒酶/端粒靶向治疗是一种潜在的有前途的癌症治疗方法，因为即使是短暂的端粒功能障碍也会诱导染色体不稳定（CIN），并可能成为肿瘤生长的障碍。然而，到目前为止，只有有限数量的针对端粒酶或端粒的化合物被确定，只有少数处于临床试验阶段。三年前，我们开发了一种双HAC测定法，可以识别和排序由于端粒功能障碍而诱导CIN的化合物。该检测基于使用两种等基因细胞系，一种携带线状HAC（含有端粒），另一种携带环状HAC（缺乏端粒）。端粒对药物治疗的破坏导致线性HAC的特异性不稳定。在最近的工作中，我们使用双HAC法分析了5种Hsp90分子伴侣抑制剂，17-AAG, TAS-116, XL888， SNX-2112和STA-9090，其中一些正在临床试验中。先前已经证明，人类Hsp90与功能性端粒酶复合物有关。出乎意料的是，所有五种伴侣抑制剂都会导致线性HAC的高损失，而对圆形HAC的稳定性没有影响。另外的实验表明，在处理过的细胞中，端粒重复序列缺乏。这意味着分析的化合物是端粒酶的特异性抑制剂，是治疗癌症的有希望的候选药物。其中一种药物TAS-116最近在日本被批准用于胃肠道间质瘤（GIST）的治疗。

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. As known, HACs are generated after transfection of alphoid DNA arrays into human cells. Efficiency of HAC formation is quite low that requires analysis of a large number of the transfectants. In our recent collaborative work, we have performed the first systematic study of structural requirements of the alphoid DNA arrays for efficient HAC formation. We demonstrated that combination of CENP-B box positive and negative synthetic alpha satellite repeats greatly improves de novo HAC formation. Previously we constructed a synthetic HAC (tetO-HAC) allowing tethering of the HAC kinetochore by different chromatin modifies fused with the Tet repressor protein. The tetO-HAC, whose centromere contains tetO repeats, has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin. In our recent study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO and lacO, i.e., a lacO/tetO-HAC. This new HAC can be used for detailed epigenetic engineering studies and to study a mechanism of centromere repositioning and inactivation in dicentric chromosomes. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. In separate experiments, a platform with multi-integrase recombination sites has been inserted into the tetO-HAC for assembly of large genetic loci in the HAC. Our work is in progress to assemble a synthetic nucleolar organizer region (NOR) in the HAC to clarify a molecular mechanism of NORs association with nucleoli. For this purpose, 45 kb rDNA units and flanking proximal junction (PJ) and distal junction (DJ) sequences were isolated from human chromosomes 21 and 22 by a transformation-associated recombination (TAR) cloning method developed in our lab. It is worth noting that analysis of the rDNA units revealed an unexpectedly high number of sequence variants in the 45S RNA region. Some variants in 18S and 28S rRNA regions may affect either ribosome RNA folding or interaction with ribosomal proteins. An entire rDNA array along with the flanking PJ and DJ regions isolated from chromosome 22 provides a platform for assembly of a synthetic NOR region. This can enable a detailed study of the sequence requirements and the mechanism of nucleolar formation in human cells, including the role of PJ and DJ in this process. To characterize the sequence requirements for nucleolar formation in human cells, we inserted different fragments of a NOR from chromosome 22 corresponding to a complete 45 kb rDNA unit, DJ and PJ regions into a gene loading site of the tetO-HAC propagated in human HT1080. To determine the nucleoli association status of the HACs carrying different TAR constructs in interphase cells, we applied 3D immune-FISH. The HACs were visualized with the PNA probe for the tetO-alphoid array. Nucleoli were visualized using an antibody against Nop52. The nucleoli association was observed for the HAC carrying the 45 kb entire rDNA repeat and the 58 kb DJ region. In contrary, the percentage of nucleoli association of the HAC carrying the 53 kb PJ fragment did not differ from the control HAC carrying the GFP transgene. Collectively, our analysis of the HACs carrying different regions of a NOR from chromosome 22 revealed that a single rDNA repeat, and a 58 kb of the DJ DNA fragment are sufficient to drive nucleolar association of these HACs. Work is in progress to clarify a molecular mechanism of NORs association with nucleoli using a HAC vector carrying different fragments of the rDNA unit and DJ region. An abnormal chromosome number is a feature of most solid tumors and is often accompanied by an elevated rate of chromosome instability (CIN). Gain or loss of entire chromosomes leads to large-scale changes in gene copy number and expression levels. Mutations in CIN genes are thought to be an early event in tumor development. At present, approximately 400 human genes that control proper chromosome transmission have been annotated with gene ontology terms, while systematic CIN gene screens in yeast have revealed more than 900 genes. Therefore, it may be supposed that many human CIN genes remain unidentified. In our previous work, we developed a high-throughput assay for identification of new human CIN genes using the tetO-HAC expressing a degron-destabilized EGFP. In the current study, we are using an available at NCATS Ambion collection of 19,000 siRNAs covering the whole human genome for screening new CIN genes. As a result, 250 new CIN candidate genes were identified. The experiments on reconfirmation of 250 CIN candidate genes are in progress. The first step is the genes knock-down using newly developed siRNAs. Identification of new CIN genes should create opportunities for the development of new therapeutic strategies to target the CIN phenotype of cancer cells. Telomerase/telomere targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. However, till now only a limited number of chemical compounds that target telomerase or telomeres have been identified and only a few are in clinical trials. Three years ago, we developed a dual HAC assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. In recent work, we used a dual HAC assay for analysis of five Hsp90 molecular chaperone inhibitors, 17-AAG, TAS-116, XL888, SNX-2112 and STA-9090, some of which are in clinical trials. It was previously demonstrated that human Hsp90 is associated with a functional telomerase complex. Unexpectedly, all five chaperone inhibitors induce a very high loss of a linear HAC with no effect on stability of a circular HAC. Additional experiments demonstrated a shortage of telomeric repeats in the treated cells. It means that the analyzed compounds are very specific inhibitors of telomerase and are promising drug candidates for treatment of cancer. One of them, TAS-116, has been recently approved for treatment of gastrointestinal stromal tumors (GIST) in Japan.