Human Artificial Chromosomes for Cancer Research and Functional Genomics

在单个NOR中，人类rDNA单位的组织和分化程度仅部分为人所知。为了解决这一缺陷，我们应用TAR克隆技术从21号和22号染色体上分离单个rDNA单元。该方法揭示了成熟的18S/28S rRNA序列出乎意料的高度异质性，提高了核糖体动力学中相应变化的可能性。这些变异在体内的功能分析是不可能的，即在外中心染色体中存在许多rRNA基因拷贝。为了克服这个障碍，我们正在使用苏珊·利布曼博士实验室开发的酵母rDNA测定法。他们构建了酵母菌株，其中染色体rDNA重复序列被完全删除，细胞生长由2微米DNA质粒上存在的单一类型的rDNA重复单元支持。利用质粒洗选，该系统可用于检测rDNA的任何突变变化对酵母细胞生长、翻译准确性以及参与核糖体组装和/或功能的rRNA结构的影响。使用该系统，我们正在测试在人类和酵母之间保守的18S和28S区域鉴定的12个序列变体。具体而言，将保守片段中的每个变体引入酵母rDNA的相应位置，并使用一个大小为9 kb的修饰完整酵母rDNA单元进行质粒改组。我们现在已经应用TAR克隆策略来表征小鼠基因组中rDNA的结构和变异。之所以选择小鼠，是因为人类疾病（包括癌症和衰老）的基因工程小鼠模型（GEMMs）已经被创造出来。尽管rDNA在小鼠中的异质性和不稳定性已经得到了充分的证明，但到目前为止，基于45S rRNA序列，只组装了一个小鼠rDNA单元和一个携带IGS区域的可用BAC。这样的非代表性参考序列排除了使用计算和生物信息学方法来识别rDNA变体。采用携带YAC和BAC磁带的TAR载体分离小鼠rDNA单元。出乎意料的是，分离克隆的大小与已发表的45 kb小鼠rDNA参考序列BK000946不对应。24个独立的tar分离的含rdna的克隆分为三个大小类别（35-40 kb， 40-45 kb和45-50 kb）。DNA测序证实了每个BAC克隆中插入物的大小。它们的序列比较表明，大小差异是由不同的IGS区域大小决定的。请注意，IGS区域的这种大小差异从未在人类和灵长类动物rDNA单位中观察到过。由于观察到的rDNA单元的大小异质性使得很难组装参考序列，我们组装了三个共识序列，即1 (38,954 bp), 2 （42,2007 bp）和3 （46,251 bp）对应于三种类型的rDNA单元。这些一致序列的比较显示了在IGS区域内sin和其他重复元件的插入和删除。相比之下，编码45S rRNA的区域相对稳定。对该区域的分析显示，tar分离的rDNA单元与已发表的“标准”45S rRNA序列之间存在206个差异。我们计划继续小鼠rDNA的表征。rDNA单元将从三种不同的小鼠品系中进行tar克隆，以阐明这些IGS大小的变异在小鼠中是否常见。最后，我们将利用实验室开发的径向TAR克隆技术来鉴定小鼠染色体中rDNA集群两侧的序列[远端（DJ）和近端（PJ）区域]。关于这些序列的信息对于分析小鼠NORs在衰老和癌变过程中的动态至关重要。哺乳动物着丝粒直接忠实的遗传，典型的特征是高度重复和快速进化的DNA区域。在最近的合作工作中，我们描述了一个小鼠物种的着丝粒创新。我们集中研究了一种名叫慕斯·帕哈里的老鼠，我们发现它的一条染色体上新进化的重复序列大量扩增，容纳了2万个功能的CENP-B盒子：比其他的M.帕哈里着丝粒多100倍。新着丝粒的亲微管和反微管结合的平衡使其能够在细胞分裂过程中与旧着丝粒分离，而旧着丝粒的序列产生了明显不同的分子组成。我们先前构建了一个合成HAC载体（tetO-HAC），它在人类着丝点的组装和维护以及基因治疗和合成生物学研究中具有很大的潜力。为了扩大tetO-HAC的效用，我们开发了两种测定染色体不稳定性（CIN）的方法。这些检测允许：i)对不同的抗癌药物和细胞毒性植物提取物对染色体传递保真度的影响进行排序；ii)鉴定可能成为癌症治疗靶点的新的CIN基因。使用基于hac的测定，我们能够对不同的抗癌药物对CIN的影响进行排序。具有不同作用机制的药物被纳入分析。通过基于hac的筛选工具鉴定出的选择性提高癌细胞中CIN的新的和潜在毒性较低的化合物可能为新的癌症治疗策略奠定基础。最近，基于hac的检测被用于评估IDH1和TP53基因中最常见的胶质瘤错义突变对染色体传播的贡献。基于此分析，IDH1R132H和TP53R248Q错义突变导致癌细胞中CIN水平升高。与传统替莫唑胺相比，我们还研究了携带这些突变的恶性胶质瘤细胞对cin诱导药物（紫杉醇、长春新碱和依托泊苷）的敏感性。本研究中发现的选择性提高CIN以促进癌细胞死亡的新的和潜在毒性较低的药物可能为胶质瘤的新治疗策略奠定基础。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，我们使用了NCATS Ambion收集的19,000个sirna，涵盖了整个人类基因组，用于鉴定新的CIN基因。结果发现了250个新的CIN候选基因。利用新开发的sirna和CRISPR敲除实验以及活细胞显微镜对250个CIN候选基因进行了再确认实验。结果，鉴定出44个新的CIN基因。实验正在进行中，以阐明这些基因在正常染色体传递中的功能。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。

The organization and the degree of divergence of the human rDNA units within an individual NOR are only partially known. To address this lacuna, we applied the TAR cloning technology to isolate individual rDNA units from chromosomes 21 and 22. This approach revealed an unexpectedly high level of heterogeneity in mature 18S/28S rRNA sequences, raising the possibility of corresponding variations in ribosome dynamics. A functional analysis of these variants in vivo, i.e., in the presence of many copies of rRNA genes in acrocentric chromosomes, is impossible. To overcome this obstacle, we are using a yeast rDNA assay developed by Dr. Susan Liebman's lab. They constructed the yeast strain, in which the chromosomal rDNA repeats were completely deleted, and cell growth was supported by the presence of a single kind of rDNA repeat units on a 2-micron DNA plasmid. Using plasmid shuffling, this system can be used to examine the effects of any mutational changes of rDNA on growth of yeast cells, on translational accuracy, and on rRNA structures involved in the ribosome assembly and/or function. Using this system, we are testing dozen sequence variants identified in 18S and 28S regions that are conservative between human and yeast. Specifically, each variant in the conservative segments is introduced to the corresponding position of yeast rDNA and a modified complete yeast rDNA unit 9 kb in size was used for plasmid shuffling. We have now applied the TAR cloning strategy to characterize the structure and variations of rDNA in the mouse genome. Mice were chosen because genetically engineered mouse models (GEMMs) of human disorders, including cancer and aging, have been created. Although rDNA heterogeneity and instability in mice are well documented, until now only one mouse rDNA unit has been assembled based on the 45S rRNA sequence and one available BAC carrying the IGS region. Such a non-representative reference sequence precludes employing computational and bioinformatic methods to identify rDNA variants. To isolate mouse rDNA units, the TAR vector carrying YAC and BAC cassettes was used. Unexpectedly, the size of the isolated clones did not correspond to the published 45 kb mouse rDNA reference sequence BK000946. 24 independent TAR-isolated rDNA-containing clones are represented by three-size classes (35-40 kb, 40-45 kb and 45-50 kb). DNA sequencing confirmed the size of inserts in each BAC clone. Their sequence comparison revealed that size difference is determined by different size of the IGS region. Note that such size variations in the IGS region have never been observed for human and primate rDNA units. Because the observed size heterogeneity of rDNA units makes it difficult to assemble a reference sequence, we assembled three consensus sequences, i.e., 1 (38,954 bp), 2 (42,007 bp) and 3 (46,251 bp) corresponding to three types of rDNA units. Comparison of these consensus sequences revealed both insertions and deletions of SINE and other repetitive elements within the IGS region. In contrast, the region encoding 45S rRNA is relatively stable. Analysis of this region revealed 206 variations between TAR-isolated rDNA units and the published "standard" 45S rRNA sequence. We plan to continue the characterization of mouse rDNA. rDNA units will be TAR-cloned from three different mouse strains to clarify if these IGS size variants are common in mice. Finally, we will employ radial TAR cloning developed in our lab to identify sequences flanking the rDNA clusters in the mouse chromosomes [distal (DJ) and proximal (PJ) regions]. Information about these sequences is critical to analyze the dynamics of mouse NORs during aging and carcinogenesis. Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. In the recent collaborative work, we described centromere innovations within a mouse species. We focused on a mouse species, Mus pahari, that we found has one chromosome with a massive expansion of a newly evolved repeat array that houses 20,000 functional CENP-B boxes: 100-fold more than on the other M. pahari centromeres. The balance of pro- and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition. We previously constructed a synthetic HAC vector (tetO-HAC) that has a great potential for the study of assembly and maintenance of human kinetochore as well as for gene therapy and synthetic biology. To extend the utility of the tetO-HAC, we have developed two assays for measuring chromosome instability (CIN). These assays allow: i) ranking different anticancer drugs and cytotoxic plant extracts with respect to their effects on chromosome transmission fidelity and ii) identifying new CIN genes that may be targets for cancer therapy. Using HAC-based assays we were able to rank different anticancer drugs with respect to their effects on CIN. Drugs with various mechanisms of action were included in the analysis. New and potentially less toxic compounds that selectively elevate CIN in cancer cells identified by the HAC-based screening tool could lay the foundation for new treatment strategies for cancer. Recently, the HAC-based assay was used to evaluate contribution of the most common glioma missense mutations in IDH1 and TP53 genes on chromosome transmission. Based on this analysis, IDH1R132H and TP53R248Q missense mutations result in a high level of CIN in cancer cells. We also investigated the sensitivity of malignant glioma cells carrying these mutations to CIN-inducing drugs - paclitaxel, vincristine, and etoposide - compared to conventional temozolomide. New and potentially less toxic agents that selectively elevate CIN to promote cancer cell death identified in this study could lay the foundation for new treatment strategies of gliomas. 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 have been used an available at NCATS Ambion collection of 19,000 siRNAs covering the whole human genome for identification of new CIN genes. As a result, 250 new CIN candidate genes have been identified. The experiments on reconfirmation of 250 CIN candidate genes have been performed using newly developed siRNAs and CRISPR knock-out experiments and live-cell microscopy. As a result, 44 new CIN genes were identified. The experiments are in progress to clarify a function of these genes in proper chromosome transmission. Identification of new CIN genes should create opportunities for the development of new therapeutic strategies to target the CIN phenotype of cancer cells.