Study of hereditary prostate cancer and human artificial chromosomes

遗传性前列腺癌与人类人工染色体的研究

• 批准号: 7733027
• 负责人: VLADIMIR LARIONOV
• 金额: $ 116.45万
• 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/7733027
• 关键词: Aneuploidy; Antigens; Architecture; Binding; Binding Sites; Boxing; Cancer Family; Cancer Patient; Cell Nucleus; Cells; Centromere; Chickens; Chromatin; Chromosomal Rearrangement; Chromosome Segregation; Chromosome Structures; Chromosomes, Artificial, Human; Classification; Code; Complex; DNA; DNA Sequence; DNA Sequence Rearrangement; Development; Disease; Distal; Ensure; Epigenetic Process; Episome; Evolution; Exhibits; Facility Construction Funding Category; Frequencies; Future; Gene Cluster; Gene Deletion; Gene Delivery; Gene Dosage; Gene Duplication; Gene Expression; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genetic Variation; Genome; Genomic Segment; Genomics; Germ Lines; Goals; Haplotypes; Heterochromatin; Histone Code; Histones; Hot Spot; Human; Human Genome; Inherited; Investigation; Kinetochores; Knowledge; Lead; Libraries; Link; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of testis; Mammalian Cell; Mediating; Methods; Mitosis; Mitotic; Mutation; Nucleic Acid Sequence Homology; Numbers; Personal Satisfaction; Polyploidy; Predisposition; Process; Proteins; Purpose; Reporting; Research; Role; Satellite DNA; Sequence Analysis; Site; Stretching; Structure; Susceptibility Gene; System; Techniques; Tetanus Helper Peptide; Tetracycline; Tetracyclines; Therapeutic; Transcription Coactivator; Transfection; Variant; Work; Yeasts; base; cancer cell; centromere autoantigen 80K; centromere protein A; centromere protein C; density; gene delivery system; gene therapy; genetic linkage; homologous recombination; human disease; in vivo; monomer; novel; novel strategies; segregation; size; sperm cell; success; synthetic construct; tool; vector

Genetic linkage studies implicate a gene or genes at Xq27 in hereditary prostate cancer susceptibility (HPCX). The corresponding region spans 750 kb and includes five SPANX genes (SPANX-A1, -A2, -B, -C, and D), which encode proteins that are expressed in sperm nuclei and a variety of cancer cells. Each SPANX gene is embedded in a recently-formed segmental duplication (SD) up to 100 kb in size, resulting in extensive enrichment in long stretches of repeated DNA in this gene region. Due to their recent amplification, both SPANX coding and flanking sequences in the SDs are nearly identical throughout the SPANX-A/D cluster, which complicates sequence analysis of these genes by PCR-based methods in the search for mutations. However, we recently succeeded in performing such an analysis of the Xq27-linked SPANX genes from prostate cancer patients, using the transformation-associated recombination (TAR) technique, which makes it possible to directly isolate large genomic segments from complex genomes. This analysis revealed frequent gene deletion/duplication and homology-based sequence transfers involving SPANX genes at Xq27, suggesting that SD-mediated homologous recombination involving the SPANX genes might lead to increased genetic instability and possibly to a higher level of genetic diversity in SPANX genes in germ lines. The results of the analysis showed that no DNA sequence variation or genetic haplotype in the SPANX gene cluster was associated with susceptibility to prostate cancer. However it remains possible that Xq27-linked prostate cancer susceptibility is related to variation in the architecture of the SPANX-A/D gene cluster. We hypothesize that X-linked predisposition to prostate cancer is caused by SD-mediated genomic rearrangements at Xq27. It is well known that SDs mediate ectopic interactions between distal chromosomal sites leading to chromosomal rearrangements such as duplications, deletions, and inversions. Such SD-mediated rearrangements can alter expression of genes in the vicinity of the SD, resulting in cellular and/or phenotypic changes and pathological disease. The density of SDs in the SPANX gene cluster is unusually high, representing more than one third of the 750 kb genomic region, suggesting a likely hot spot for genomic rearrangements. Based on the structure and organization of duplicated segments at Xq27, a high frequency of deletions and duplications of SPANX genes and flanking DNA as well as inversions of SPANX-containing regions is predicted. Some of them may result in altered expression of one or more genes within or near the SPANX gene cluster. Therefore, future work will focus on identifying inversion(s) within the SPANX-A/D gene cluster in X-linked prostate cancer families, which could lead to malignancy. After more than two decades of investigation, human centromeres remain enigmatic and poorly understood. Some progress in this field was outlined after demonstration by Hunt Willards group that alpha satellite DNA (alphoid DNA), the primary DNA found in human centromeres, can induce the seeding of a kinetochore complex in human HT1080 cells. Several groups have confirmed this observation and reported the formation of Human Artificial Chromosomes (HACs) in human cells, using a transfection strategy that involved alpha satellite DNA. These HACs are maintained as single copy episomes (i.e., copy number of 1) in the nucleus and have a fully functional kinetochore. The development and detailed studies of HACs offer new approaches for: 1) elucidating the mechanisms for de novo centromere/kinetochore formation and its structural/functional organization, and 2) creating gene delivery vectors with potential therapeutic applications. Until recently, HACs were constructed from 50-100 kb alphoid DNA fragments identified in existing YAC or BAC libraries. As a rule, the complete DNA sequence of these fragments was unknown, which did not allow making a final conclusion regarding the structural requirements for de novo kinetochore formation. Following the establishment of our unit in NCI, we have focused part of our research on constructing synthetic alphoid DNA arrays with precisely-defined DNA sequence variations. For this purpose, we developed a novel method for construction of alphoid DNA arrays, CADA, exploiting in vivo recombination in yeast. This method represents a tool for mutational analysis of alphoid DNA, as each of the constructed arrays can be evaluated for its ability to form a HAC and if necessary can be modified. Knowledge of combinatory of DNA sequences initiating HAC formation from alphoid DNA substrates may provide unique information on the functional organization of the mammalian centromere. It may also accelerate the construction of a more sophisticated HAC-based system for gene delivery and expression. Among the main unsolved problems of the HAC system are: i) a low efficiency of de novo HAC formation, and ii) multimerization of the input DNA concomitant with a HAC formation, making it difficult to control gene copy number and the location of genes in a HAC. Optimization of the centromeric component of HAC constructs is likely to be key to ensuring an efficient seeding of a functional kinetochore, and may also influence the extent of DNA multimerization during HAC formation. Using CADA method, we have recently determined the minimal size of alphoid DNA array capable of forming a HAC and constructed a novel HAC to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric alpha-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tetracycline repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused mis-segregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C and H3K4me2 from the centromere, accompanied by an accumulation of histone H3K9me3. In addition to providing the first clear demonstration that heterochromatin within the centromere is incompatible with kinetochore activity, the conditional centromere of the HAC opens a new spectrum of opportunities for the systematic manipulation of the histone code within the kinetochore, and definition of the full epigenetic signature of centromeric chromatin. The new HAC with a conditional centromere also has potential as a system for gene delivery and regulated gene expression in mammalian cells. To make possible a regulated gene expression in HACs, the tet-O containing HAC was transferred from human host cells into chicken DT40 cells exhibiting a high level of homologous recombination. Our recent studies show that the HAC with a conditional centromere has high mitotic stability and is maintained in a single copy in DT40 cells. To generate a HAC-based system for expression of mammalian genes, a unique lox-P was introduced into the HAC with a conditional centromere using a recombinational machinery of DT [summary truncated at 7800 characters]

遗传连锁研究表明 Xq27 上的一个或多个基因与遗传性前列腺癌 (HPCX) 易感性有关。相应的区域跨越 750 kb，包括五个 SPANX 基因（SPANX-A1、-A2、-B、-C 和 D），它们编码在精子细胞核和各种癌细胞中表达的蛋白质。每个 SPANX 基因都嵌入最近形成的大小高达 100 kb 的片段重复 (SD) 中，从而导致该基因区域中长段重复 DNA 的广泛富集。由于最近的扩增，SD 中的 SPANX 编码和侧翼序列在整个 SPANX-A/D 簇中几乎相同，这使得在寻找突变时通过基于 PCR 的方法对这些基因进行序列分析变得复杂。 然而，我们最近使用转化相关重组（TAR）技术成功地对前列腺癌患者的 Xq27 连接的 SPANX 基因进行了这样的分析，这使得从复杂基因组中直接分离大基因组片段成为可能。该分析揭示了涉及 Xq27 处 SPANX 基因的频繁基因删除/重复和基于同源性的序列转移，表明涉及 SPANX 基因的 SD 介导的同源重组可能导致遗传不稳定性增加，并可能导致种系中 SPANX 基因的遗传多样性水平更高。分析结果表明，SPANX基因簇中的DNA序列变异或遗传单倍型与前列腺癌易感性无关。然而，Xq27 连锁的前列腺癌易感性仍然可能与 SPANX-A/D 基因簇结构的变异有关。我们假设 X 连锁前列腺癌易感性是由 SD 介导的 Xq27 基因组重排引起的。众所周知，SDs 介导远端染色体位点之间的异位相互作用，导致染色体重排，例如重复、缺失和倒位。这种 SD 介导的重排可以改变 SD 附近基因的表达，导致细胞和/或表型变化和病理疾病。 SPANX 基因簇中 SD 的密度异常高，占 750 kb 基因组区域的三分之一以上，表明可能是基因组重排的热点。根据 Xq27 重复片段的结构和组织，预测 SPANX 基因和侧翼 DNA 的高频率缺失和重复以及包含 SPANX 的区域的倒位。其中一些可能会导致 SPANX 基因簇内或附近的一个或多个基因的表达发生改变。因此，未来的工作将集中于识别 X 连锁前列腺癌家族中 SPANX-A/D 基因簇内的倒位，这可能导致恶性肿瘤。经过二十多年的研究，人类着丝粒仍然是个谜，人们对其知之甚少。 Hunt Willards 小组证明 α 卫星 DNA（alphoid DNA）（人类着丝粒中发现的主要 DNA）可以诱导人类 HT1080 细胞中动粒复合体的播种，随后概述了该领域的一些进展。几个研究小组已经证实了这一观察结果，并报告了使用涉及 α 卫星 DNA 的转染策略在人类细胞中形成了人类人工染色体 (HAC)。这些 HAC 在细胞核中保持为单拷贝附加体（即拷贝数为 1），并具有功能齐全的动粒。 HAC 的开发和详细研究为以下方面提供了新方法：1）阐明着丝粒/着丝粒从头形成的机制及其结构/功能组织，2）创建具有潜在治疗应用的基因传递载体。直到最近，HAC 都是由现有 YAC 或 BAC 文库中鉴定的 50-100 kb alphoid DNA 片段构建的。 通常，这些片段的完整 DNA 序列是未知的，因此无法就动粒从头形成的结构要求得出最终结论。在 NCI 成立我们的部门后，我们的部分研究重点是构建具有精确定义的 DNA 序列变异的合成 alphoid DNA 阵列。为此，我们开发了一种构建阿尔法 DNA 阵列的新方法，即 CADA，利用酵母体内的重组。该方法代表了一种用于 alpoid DNA 突变分析的工具，因为可以评估每个构建的阵列形成 HAC 的能力，并在必要时进行修改。了解从类 DNA 底物启动 HAC 形成的 DNA 序列组合可以提供有关哺乳动物着丝粒功能组织的独特信息。它还可能加速构建更复杂的基于 HAC 的基因传递和表达系统。 HAC系统尚未解决的主要问题包括：i）从头HAC形成的效率低，以及ii）伴随HAC形成的输入DNA的多聚化，使得难以控制基因拷贝数和基因在HAC中的位置。 HAC 构建体着丝粒成分的优化可能是确保功能性动粒有效接种的关键，并且还可能影响 HAC 形成过程中 DNA 多聚化的程度。使用CADA方法，我们最近确定了能够形成HAC的最小尺寸的αDNA阵列，并构建了一种新型HAC来操纵活跃着丝粒内染色质的表观遗传状态。 HAC 具有二聚体 α 卫星重复序列，其中包含一种具有 CENP-B 结合位点的天然单体，以及一种完全人工合成的单体，其中 CENP-B 盒被四环素操纵子 (tetO) 取代。该 HAC 表现出正常的着丝粒蛋白组成和有丝分裂稳定性。将多个四环素阻遏物 (tetR) 融合物靶向着丝粒对着丝粒功能没有影响。然而，使用 tTA 转录激活子将染色质状态改变为更开放的配置，或使用 tTS 转录沉默子将染色质状态改变为更封闭的状态，会导致 HAC 的错误分离和丢失。 tTS 结合导致着丝粒中 CENP-A、CENP-B、CENP-C 和 H3K4me2 的丢失，并伴有组蛋白 H3K9me3 的积累。除了首次清楚地证明着丝粒内的异染色质与着丝粒活性不相容外，HAC 的条件着丝粒还为系统操纵着丝粒内的组蛋白密码以及定义着丝粒染色质的完整表观遗传特征开辟了新的机会。具有条件着丝粒的新 HAC 也具有作为哺乳动物细胞中基因传递和调节基因表达的系统的潜力。为了使 HAC 中的基因表达受调控成为可能，将含有 Tet-O 的 HAC 从人宿主细胞转移到鸡 DT40 细胞中，表现出高水平的同源重组。我们最近的研究表明，具有条件着丝粒的 HAC 具有较高的有丝分裂稳定性，并且在 DT40 细胞中维持单拷贝。为了生成基于 HAC 的哺乳动物基因表达系统，使用 DT 重组机制将独特的 lox-P 引入具有条件着丝粒的 HAC [摘要截断为 7800 个字符]

项目成果

Accelerated evolution of the ASPM gene controlling brain size begins prior to human brain expansion.

• DOI: 10.1371/journal.pbio.0020126
• 发表时间: 2004-05
• 期刊: PLoS biology
• 影响因子: 9.8
• 作者: Kouprina N;Pavlicek A;Mochida GH;Solomon G;Gersch W;Yoon YH;Collura R;Ruvolo M;Barrett JC;Woods CG;Walsh CA;Jurka J;Larionov V
• 通讯作者: Larionov V

Inactivation of a human kinetochore by specific targeting of chromatin modifiers.

• DOI: 10.1016/j.devcel.2008.02.001
• 发表时间: 2008-04
• 期刊: DEVELOPMENTAL CELL
• 影响因子: 11.8
• 作者: Nakano, Megumi;Cardinale, Stefano;Noskov, Vladimir N.;Gassmann, Reto;Vagnarelli, Paola;Kandels-Lewis, Stefanie;Larionov, Vladimir;Earnshaw, William C.;Masumoto, Hiroshi
• 通讯作者: Masumoto, Hiroshi

Exploring transformation-associated recombination cloning for selective isolation of genomic regions.

探索转化相关重组克隆以选择性分离基因组区域。

• DOI: 10.1385/1-59259-752-1:069
• 发表时间: 2004
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Kouprina,Natalay;Noskov,VladimirN;Koriabine,Maxim;Leem,Sun-Hee;Larionov,Vladimir
• 通讯作者: Larionov,Vladimir