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: 10262084
• 负责人: VLADIMIR LARIONOV
• 金额: $ 222.93万
• 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/10262084
• 关键词: Address; Affect; Aneuploidy; Antineoplastic Agents; Bacterial Artificial Chromosomes; Biological Assay; Biotechnology; Catalogs; Cell Cycle Regulation; Cell Line; Cell division; Cells; Centromere; Characteristics; Chromatin; Chromosomal Instability; Chromosome 21; Chromosome 22; Chromosome Segregation; Chromosomes; Chromosomes, Artificial, Human; Cloning; Collection; Cytology; DNA Damage; Development; Diamond-Blackfan anemia; Disease; Distal; Epigenetic Process; Equilibrium; Ethnic group; Evolution; F8 gene; Flow Cytometry; Fluorescence; Frequencies; Functional disorder; G-Quartets; Gene Delivery; Gene Duplication; Gene Expression; Generations; Genes; Genetic Diseases; Genetic Recombination; Genetic study; Genome; Genomic DNA; Genomics; Goals; Hemophilia A; Heterochromatin; Heterogeneity; Human; Human Chromosomes; Human Genome; Image; Individual; Inherited; Integrase; Kinetochores; Knowledge; Lead; Libraries; Ligands; Maintenance; Malignant Neoplasms; Measurement; Measures; Messenger RNA; Methods; Microtubule Stabilization; Microtubule stabilizing agent; Mitosis; Mitotic; Modeling; Molecular; Mus; Nucleolar Organizer Region; Ontology; Pathology; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Polyploidy; Process; Protein Kinase; Proteins; Protocols documentation; Reporting; Repressor Proteins; Research; Ribosomal DNA; Ribosomes; Role; Satellite DNA; Severities; Site; Small Interfering RNA; Structure; TOP1 gene; Tandem Repeat Sequences; Telomerase; Telomere Maintenance; Tetanus Helper Peptide; Therapeutic; Topoisomerase Inhibitors; Transgenes; Translations; Variant; Work; Yeasts; anticancer research; base; cancer cell; cancer therapy; cancer type; centromere autoantigen 80K; centromere protein A; chromosome loss; chromosome missegregation; design; epigenetic memory; functional genomics; gene therapy; genetic variant; induced pluripotent stem cell; insight; novel; novel therapeutic intervention; novel therapeutics; rRNA Genes; response; screening; synthetic biology; telomere; transcription factor; transmission process; tumor; tumor growth; tumor progression; vector

HACs assembled from alpha-satellite 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, synthetic biology, 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 a role of different types of chromatin in kinetochore function. During the past year, the same approach exploiting the tethering of the tetO-HAC kinetochore revealed that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory. We also demonstrated that CENP-B protein balances assembly of CENP-A centrochromatin or heterochromatin formation on satellite DNA. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of kinetochore. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. Previously, we have demonstrated functional expression of dozen genes loaded into the alphoidtetO-HAC. In recent study, we have developed a gene therapy model that employs the teO-HAC as a gene delivery vector in the treatment of the monogenic genetic disease hemophilia A in mice. It was demonstrated that the therapeutic HAC is maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. For gene expression/function analyses, a platform with multi-integrase recombination sites has been inserted into tetO-HAC. This platform allows assembly of unlimited number of genomic DNA segments in the HAC. Work is in progress to use this HAC for assembling of synthetic nucleolar organizer region (NOR) from human rDNA units and flanking regions recently isolated and characterized in our lab. It worth noting that despite the key role of rRNA genes, little is known about the extent of sequence variation in ribosomal DNA (rDNA). The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of human rDNA units within an individual NOR are only partially known. To address this lacuna, we first applied TAR cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Additional studies are required to clarify if some of the reported variations have functional consequences - for example, speculatively affecting the assembly of ribosomes; the selection or efficiency of translation of particular mRNAs; and/or the severity of "ribosomopathies" like Diamond-Blackfan anemia. It is worth noting that this is the first example of isolation of s YAC/BAC clones corresponding to rDNA array and long flanking sequences corresponding to PJ and DJ regions. tetO-HAC with multi-integrase recombination sites opens the opportunity to assemble the entire NOR of the chromosome 22. Such a HAC module that is transferable into any type of human cells will be used to investigate the role of NORs in nucleolar formation and function, and can initiate studies of the role of these regions in the well-known empirical association of nucleoli with pathology. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. 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 300 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. In recent study the HAC-based assay was applied for analysis of newly developed microtubule-stabilizing compounds. The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In our recent work, we designed a new protocol to screen for, and rank the efficacy of, compounds specifically targeting telomeres and telomerase. The protocol is based on the use of two isogenic cell lines containing a circular HAC (lacking telomeres) and a linear HAC (containing telomeres): compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, we found several compounds (derivatives of Pt ttpy) that induce a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Cytological analysis showed that chromosome loss after drugs treatment correlated with the induction of telomere-associated DNA damage. Identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction 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 because not all genes controlling chromosome transmission have yet been identified. Indeed, 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. To address this problem, we have developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses tetO-HAC expressing the GFP transgene. When this assay was applied to screen a siRNA library of protein kinases we identified 6 new genes whose deletion promotes CIN. At present, the HAC-based assay is applied for screening the Ambion collection of different siRNAs libraries (cell cycle regulation, DNA damage response, epigenetics, transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells. .

由α -卫星DNA阵列组装的HACs代表了一种新的载体，在研究人类着丝点的组装和维护以及基因治疗、合成生物学、抗癌药物筛选和生物技术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。这种HAC被成功地用于阐明不同类型的染色质在着丝点功能中的作用。在过去的一年里，同样的方法利用tetO-HAC着丝点的系结揭示了最低水平的异染色质是稳定有丝分裂着丝粒功能所必需的，但不需要维持着丝粒表观遗传记忆。我们还证明了CENP-B蛋白平衡了卫星DNA上CENP-A中染色质或异染色质形成的组装。与其他HAC载体相比，tetO-HAC具有优势，因为它可以很容易地通过使着丝点失活而从细胞中消除。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。在此之前，我们已经证明了装载到alphoidtetO-HAC中的十几种基因的功能表达。在最近的研究中，我们开发了一种基因治疗模型，利用teO-HAC作为基因传递载体治疗小鼠单基因遗传病血友病a。结果表明，在小鼠iPSCs中，治疗性HAC作为一种非整合载体保持存在，显示出组成性FVIII表达。为了进行基因表达/功能分析，在tetO-HAC中插入了一个具有多整合酶重组位点的平台。该平台允许在HAC中组装无限数量的基因组DNA片段。利用这种HAC从我们实验室最近分离和表征的人类rDNA单元和侧翼区域组装合成核仁组织者区（NOR）的工作正在进行中。值得注意的是，尽管rRNA基因发挥了关键作用，但人们对核糖体DNA （rDNA）序列变异的程度知之甚少。人类染色体13、14、15、21和22上的rDNA簇和侧翼序列代表了当前基因组组装的巨大缺口。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，我们首先应用TAR克隆技术从21号染色体上分离单个rDNA单元。该方法揭示了人类rDNA中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。我们现在已经应用同样的策略来分析整个rDNA阵列端到端的22号染色体拷贝。TAR分离物的测序提供了整个NOR序列，包括可能参与核仁功能的近端和远端连接。将新测序的rDNA与参考序列进行比较，发现在不同种族群体的个体中，人类rDNA中存在共同的变异，其中许多变异频率很高。需要进一步的研究来澄清所报道的一些变异是否具有功能性后果-例如，推测性地影响核糖体的组装；特定mrna的选择或翻译效率；和/或“核糖体病”的严重程度，如Diamond-Blackfan贫血。值得注意的是，这是首次分离到与rDNA阵列对应的5个YAC/BAC克隆和与PJ和DJ区对应的长侧翼序列。具有多整合酶重组位点的tetO-HAC为组装22号染色体的整个NOR提供了机会。这种可转移到任何类型人类细胞的HAC模块将用于研究NORs在核仁形成和功能中的作用，并可以启动这些区域在众所周知的核仁与病理的经验关联中的作用的研究。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）。CIN表现为细胞分裂时染色体分布不均匀，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300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。在最近的研究中，基于hac的分析方法被应用于新开发的微管稳定化合物的分析。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，我们设计了一种新的方案来筛选，并对专门针对端粒和端粒酶的化合物的功效进行排名。该方案基于使用两种等基因细胞系，其中含有环状HAC（缺乏端粒）和线性HAC（含有端粒）：靶向端粒酶或端粒的化合物应优先诱导线性HAC而不是环状HAC的损失。我们应用这种双hac测定法对一组已知和新开发的化合物进行排序，包括g -四联体（G4）配体。在后一组中，我们发现了几种化合物（Pt ttpy的衍生物），它们诱导线状HAC的高损失率，但对圆形HAC的有丝分裂稳定性没有显著影响。细胞学分析表明，药物治疗后染色体丢失与端粒相关DNA损伤的诱导相关。鉴定和排序由于端粒功能障碍而大大增加染色体错分离率的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的分子基础仍然未知，部分原因是并非所有控制染色体传递的基因都尚未确定。事实上，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论术语进行了注释，而在酵母中系统的CIN基因筛选已经揭示了900多个基因。为了解决这个问题，我们开发了一种实验性高通量成像（HTI） siRNA分析，可以识别新的CIN基因。我们的方法使用表达GFP转基因的tetO-HAC。当这个实验应用于筛选蛋白激酶的siRNA文库时，我们发现了6个新的基因，它们的缺失促进了CIN。目前，基于hac的检测方法被用于筛选Ambion收集的不同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。对CIN基因完整谱的鉴定将揭示染色体分离机制的新见解，并可能加快针对癌细胞中CIN表型的新治疗策略的发展。