Human Artificial Chromosomes for Cancer Research and Functional Genomics

用于癌症研究和功能基因组学的人类人工染色体

• 批准号: 10014366
• 负责人: VLADIMIR LARIONOV
• 金额: $ 182.17万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014366
• 关键词: Address; Alleles; Aneuploidy; Antineoplastic Agents; Artificial Chromosomes; Bacterial Artificial Chromosomes; Binding; Biological Assay; Biology; Biotechnology; Bypass; Catalogs; Cell Cycle Regulation; Cell Proliferation; Cell division; Cells; Cellular Stress; Centromere; Characteristics; Chromatin; Chromatin Remodeling Factor; Chromosomal Instability; Chromosome Segregation; Chromosomes; Chromosomes, Artificial, Human; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 22; Cloning; Cytology; DNA; DNA Damage; Development; Disease; Distal; Epigenetic Process; Evolution; Flow Cytometry; Fluorescence; Frequencies; Gene Duplication; Generations; Genes; Genetic Recombination; Genetic Transcription; Genetic study; Genome; Genomic DNA; Genomics; Goals; Human; Human Chromosomes; Human Genome; Individual; Inherited; Integrase; Kinetochores; Knowledge; Lead; Libraries; Location; Maintenance; Malignant Neoplasms; Measurement; Measures; Microtubule stabilizing agent; Mitosis; Molecular; Nucleolar Organizer Region; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiology; Polyploidy; Positioning Attribute; Process; Property; Protein Biosynthesis; Protein Kinase; Reporting; Repressor Proteins; Research; Ribosomal DNA; Ribosomal RNA; Ribosomes; Role; Satellite DNA; Site; Small Interfering RNA; Structure; System; TOP1 gene; Tandem Repeat Sequences; Telomerase; Telomere Maintenance; Tetanus Helper Peptide; Therapeutic; Topoisomerase Inhibitors; Transgenes; Variant; Work; Yeasts; anticancer research; base; biological adaptation to stress; cancer cell; cancer therapy; cancer type; chromosome loss; experimental study; functional genomics; gene therapy; genetic variant; human disease; induced pluripotent stem cell; novel; novel therapeutic intervention; rRNA Precursor; response; screening; small molecule libraries; structural genomics; synthetic biology; targeted cancer therapy; telomere; transcription factor; transmission process; tumor; tumor growth; tumor progression; vector

Human Artificial Chromosomes (HACs) assembled from alpha-satellte 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. 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 or tTA, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. We demonstrated that this HAC can be transferred and maintained in human iPSCs as autonomous chromosome without effecting pluripotent properties of the cells. 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. A new tetO-HAC system has several notable advantages that set it apart from other artificial chromosome-based systems, including assembly of unlimited number of genomic DNA segments and the opportunity to remove mis-incorporated DNA segments. Work is in progress to use multi-integrase system for assembling of synthetic nucleolar organizer region (NOR) in the HAC from human rDNA units isolated by transformation-associated recombination (TAR). Such a HAC module 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. Note that despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. In our recent work, we recovered ribosomal DNA segments from a single human acrocentric chromosome (chromosome 21) using TAR cloning in yeast. Accurate long-read sequencing of TAR-isolated units from the chromosome revealed substantial variation among tandem repeat rDNA copies and several palindromic structures. These clones revealed few hundred variant positions in the 45S transcription unit and in the intergenic spacer sequence. The large number of variants observed provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human diseases. Even after the completion of the human genome sequence few rDNA-containing BAC clones were sequenced and the genomic structure of entire rDNA clusters on human chromosomes 13, 14, 15, 21 and 22 are still unknown and these regions represent large gaps in the current genomic assembly. So far we do not know organization and divergence of rDNA units within individual NORs. To address this question, during the past year, we are focused on analysis of an individual NOR using TAR cloning strategy developed in our lab. This work resulted in assembly of the entire NOR sequence on human chromosome 22 along with long flanking proximal and distal sequences. Thus, we succeeded to close one of the gaps on acrocentric chromosomes. The loading of different variants of a human rDNA unit identified in our study into the tetO-HAC may help to clarify the peculiarity of PolI transcription machinery as well as the mechanism by which rDNA units are selected and targeted for chromatin changes leading to heterochromatinization in NORs. This work is now in progress. 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 themfrom 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 200 anticancer drugs on their effect on HAC loss. The strongest effect was observed for microtubule-stabilizing agents and inhibitors of topoisomerase TOP1, developed in our branch. The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In our recent work, we applied our HAC system to screen for, and rank the efficacy of, compounds specifically targeting telomeres and telomerase. The study revealed dozen compounds that selectively target telomerase or telomeres. Cytological analysis showed that chromosome instability after the drug treatment correlated with the induction of telomere-associated DNA damage. New compounds that greatly increase CIN may expedite the development of new therapeutic strategies for cancer treatment. In the vast majority of human tumors the molecular basis of CIN remains unknown, partially due to not all genes controlling proper chromosome transmission having been identified yet. We demonstrated the utility of the HAC-based assay for identification of new genes controlling chromosome transmission in human cells. 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 new six CIN genes with no previous information on their role in chromosome transmission. This assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, transcription factors) to identify other genes involved in CIN. Each of these new CIN genes may be considered as a new target for cancer therapy. During the past year, we also worked on the development of a new strategy to built HACs with a functional kinetochore. All prior HACs required large arrays of alpha-satellite DNA repeats for their construction. Bypassing of this rule would have several clear benefits. First, HAC construction would be greatly facilitated. Traditional constructs for HAC formation contain 50-200 kb of highly repetitive DNA complicating handling at all steps, from their initial construction to their clonal stability during bacterial propagation. Second, mapping the chromatin features of HACs using sequencing-based approaches would become possible. In our recent work, we report the development of a type of HAC that functions independently of these constraints. Specifically, we demonstrated that human chromosomal regions corresponding to neocentromeres are competent in de novo kinetochore formation. Importantly, that these regions contain no any centromeric repeats. Thus our study reveals molecular requirements for centromere establishment and demonstrates that alpha-satellite DNA can be bypassed altogether, thereby greatly facilitating the construction of HACs and expending the toolbox for centromere biology studies, gene therapy applications and synthetic biology efforts.

由α -卫星DNA阵列组装而成的人类人工染色体（Human Artificial chromosome, HACs）是一种新型载体，在研究人类着丝点的组装和维持、基因治疗、抗癌药物筛选和生物技术等方面具有很大的潜力。我们之前构建了一个合成的HAC (tetO-HAC)，允许通过与tet抑制蛋白融合的不同染色质修饰来系住其着丝点。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或tTA）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。诱导HAC丢失的机会为加载到tetO-HAC中的基因诱导的表型提供了独特的控制。我们证明了这种HAC可以作为自主染色体在人类iPSCs中转移和维持，而不影响细胞的多能性。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，并成功地用于HAC中的基因组装。新的tetO-HAC系统有几个显著的优势，使其与其他基于人工染色体的系统不同，包括无限数量的基因组DNA片段的组装和去除错误结合的DNA片段的机会。利用多整合酶系统从转化相关重组（TAR）分离的人类rDNA单元组装HAC中的合成核仁组织区（NOR）的工作正在进行中。该HAC模块将用于研究rDNA重复序列对核核位置的要求以及rDNA单位拷贝数对细胞增殖和应激反应的影响。值得注意的是，尽管人类核糖体在蛋白质生物合成中发挥着关键作用，但人们对核糖体DNA （rDNA）及其pre-rRNA和rRNA产物的序列变异程度知之甚少。在我们最近的工作中，我们利用酵母的TAR克隆技术从单个人类顶中心染色体（21号染色体）中恢复了核糖体DNA片段。从染色体上对tar分离的单位进行精确的长读测序，发现串联重复rDNA拷贝和几个回文结构之间存在实质性差异。这些克隆在45S转录单元和基因间间隔序列中发现了几百个变异位点。观察到的大量变异为探索基因组编码变异rRNA等位基因的表达导致物理和功能异质核糖体的可能性提供了一个关键框架，这些核糖体有助于哺乳动物生理和人类疾病。即使在人类基因组序列完成后，很少有含有rDNA的BAC克隆被测序，人类染色体13、14、15、21和22上的整个rDNA簇的基因组结构仍然未知，这些区域在目前的基因组组装中存在很大的空白。到目前为止，我们还不知道单个NORs中rDNA单元的组织和分化。为了解决这个问题，在过去的一年中，我们专注于使用我们实验室开发的TAR克隆策略分析单个NOR。这项工作的结果是在人类22号染色体上组装了整个NOR序列以及长侧翼的近端和远端序列。因此，我们成功地关闭了顶心染色体上的一个间隙。在我们的研究中，将人类rDNA单元的不同变体装载到tetO-HAC中，可能有助于阐明PolI转录机制的特殊性，以及rDNA单元被选择和靶向导致NORs异染色质化的染色质变化的机制。这项工作目前正在进行中。我们还应用我们的tetO-HAC来测量人类细胞的染色体不稳定性（CIN）。全染色体不稳定性（CIN）表现为细胞分裂过程中染色体分布不均匀，是大多数类型癌症的特征，从而将其与正常肿瘤区分开来。尽管CIN通常被认为是肿瘤生长的驱动因素，但存在一个阈值水平，使得CIN频率的进一步增加成为肿瘤生长的障碍，因此可以用于治疗。然而，目前已知能使CIN升高超过这一治疗阈值的药物数量很少。在我们之前的工作中，我们开发了一种新的定量检测方法，用于测量CIN，该方法基于使用非必需HAC携带组成型表达EGFP转基因。因此，继承HAC的细胞显示绿色荧光，而缺乏HAC的细胞则没有。这允许通过常规流式细胞术测量药物治疗反应的HAC损失率。我们用这种方法对200多种抗癌药物对HAC损失的影响进行了排名。微管稳定剂和拓扑异构酶TOP1抑制剂的效果最强。端粒酶的靶向和端粒维持机制代表了一种有希望的治疗各种类型癌症的方法。在我们最近的工作中，我们应用我们的HAC系统来筛选特异性靶向端粒和端粒酶的化合物，并对其功效进行排序。该研究揭示了十几种选择性靶向端粒酶或端粒的化合物。细胞学分析显示，药物治疗后染色体不稳定与端粒相关DNA损伤的诱导相关。大大增加CIN的新化合物可能会加速癌症治疗新治疗策略的发展。在绝大多数人类肿瘤中，CIN的分子基础仍然是未知的，部分原因是并非所有控制适当染色体传递的基因都已被确定。我们展示了基于hac的测定方法用于鉴定控制人类细胞中染色体传递的新基因的实用性。在我们最近的工作中，我们对EGFP-HAC进行了改进，并将原来的检测方法转化为人类基因化学文库和siRNA文库的高通量CIN筛选。针对720个人类蛋白激酶基因的sirna分析揭示了6个新的CIN基因，这些基因在染色体传播中没有任何信息。该方法可用于筛选不同的siRNA文库（细胞周期调控、DNA损伤反应、表观遗传学、转录因子），以鉴定参与CIN的其他基因。这些新的CIN基因中的每一个都可能被认为是癌症治疗的新靶点。在过去的一年里，我们也致力于开发一种新的策略来构建具有功能着丝点的HACs。所有先前的HACs都需要大量的α -卫星DNA重复序列来构建。绕过这条规则将有几个明显的好处。第一，将大大便利HAC的建设。传统的HAC构建包含50-200 kb的高度重复DNA，从最初的构建到细菌繁殖过程中的克隆稳定性，所有步骤的处理都很复杂。其次，利用基于测序的方法绘制HACs的染色质特征将成为可能。在我们最近的工作中，我们报告了一种独立于这些约束的HAC的发展。具体来说，我们证明了与新着丝粒相对应的人类染色体区域能够重新形成着丝粒。重要的是，这些区域不包含任何着丝粒重复序列。因此，我们的研究揭示了着丝粒建立的分子要求，并证明了α -卫星DNA可以完全绕过，从而极大地促进了HACs的构建，扩展了着丝粒生物学研究、基因治疗应用和合成生物学工作的工具箱。