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]

遗传连锁研究暗示XQ27的基因或基因在遗传性前列腺癌敏感性（HPCX）中。相应的区域跨度为750 kb，包括五个Spanx基因（Spanx -A1，-a2，-b，-c和D），它们编码在精子核和多种癌细胞中表达的蛋白质。每个Spanx基因都嵌入到近期成型的分段重复（SD）中，大小高达100 kb，从而在该基因区域的长期重复DNA中富集了广泛的富集。由于它们最近的扩增，SPS中的Spanx编码和侧翼序列在整个SPANX-A/D群集中几乎相同，这在搜索突变时通过基于PCR的方法使这些基因的序列分析变得复杂。 但是，我们最近使用与转化相关的重组（TAR）技术对来自前列腺癌患者的XQ27连接Spanx基因进行了这种分析，这使得可以将大型基因组段与复杂基因组直接分离。该分析表明，涉及XQ27处SPANX基因的基因缺失/重复和基于同源性的序列转移，这表明涉及Spanx基因的SD介导的同源重组可能会导致遗传不稳定增加，并且可能导致细菌线中Spanx基因的遗传多样性的较高水平。分析的结果表明，Spanx基因簇中没有DNA序列变异或遗传单倍型与前列腺癌的敏感性有关。但是，XQ27连接的前列腺癌敏感性仍然与Spanx-A/D基因簇的结构变化有关。我们假设前列腺癌的X连锁倾向是由SD介导的基因组重排引起的XQ27。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，缺失和反转。这种SD介导的重排可以改变SD附近基因的表达，从而导致细胞和/或表型变化和病理疾病。 Spanx基因簇中的SDS密度异常高，代表了750 Kb基因组区域中的三分之一以上，这表明可能是基因组重排的热点。根据XQ27重复段的结构和组织，可以预测Spanx基因和侧翼DNA的高频率和重复的频率以及含Spanx的区域的反演。其中一些可能导致Spanx基因簇内或附近一个或多个基因的表达改变。因此，未来的工作将集中在X连锁前列腺癌家族中SPANX-A/D基因簇内的反转，这可能导致恶性肿瘤。经过二十多年的调查，人类的centromeres仍然神秘且理解不足。亨特·威拉德斯（Hunt Willards）组示威表明，在人centromeres中发现的主要DNAα卫星DNA（Alphoid DNA）可以诱导人类HT1080细胞中的Kinetochore复合物的播种。几个小组已经证实了这一观察结果，并使用涉及α卫星DNA的转染策略报告了人类细胞中人造染色体（HAC）的形成。这些HAC在核中保持为单个拷贝发作（即，拷贝数为1），并具有功能齐全的动力学。 HACS的开发和详细研究为以下方法提供了新方法：1）阐明新的Centromere/Kinetochore组的机制及其结构/功能组织，以及2）创建具有潜在治疗应用的基因递送向量。直到最近，HACS是在现有YAC或BAC文库中鉴定出的50-100 kb字母DNA片段中构建的。 通常，这些片段的完整DNA序列是未知的，这不允许对从头运动学形成的结构要求得出最终结论。在NCI中建立单元后，我们将一部分研究集中在构建具有精确定义的DNA序列变化的合成字母DNA阵列上。为此，我们开发了一种新颖的方法，用于构建字母DNA阵列，CADA，在酵母中利用体内重组。该方法代表了用于对字段性DNA的突变分析的工具，因为可以评估每个构造的阵列形成HAC的能力，并在必要时进行修改。了解从字段性DNA底物启动HAC形成的DNA序列组合的知识可能会提供有关哺乳动物centromere功能组织的独特信息。它还可以加速基于HAC的基因递送和表达的更复杂的系统。 HAC系统的主要未解决问题之一是：i）从头形成的效率低，ii）输入DNA与HAC形成的多个多次化，使得难以控制基因拷贝数和HAC中基因的位置。 HAC构建体的中心粒分量的优化可能是确保功能性动力学的有效播种的关键，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并构建一种新型HAC来操纵活性动力学内染色质的表观遗传状态的字母DNA阵列的最小尺寸。 HAC具有一个二聚体α-卫星重复，该重复含有一个带有CENP-B结合位点的天然单体，而一个完全人造的合成单体，其CENP-B盒子被Tetracycline Operator（TETO）代替。该HAC表现出正常的动力学蛋白组成和有丝分裂稳定性。将几个四环素阻遏物（TER）融合到丝粒中对动力学函数没有影响。但是，将染色质状态更改为使用TTA转录激活因子或更封闭状态的更开放的配置，而TTS转录消音器会导致HAC的错误分离和丢失。 TTS结合导致CENP-A，CENP-B，CENP-C和H3K4ME2的损失，伴随组蛋白H3K9Me3的积累。除了提供第一个明确的证明外，丝粒内的异染色质与动力学活性不相容，HAC的条件性丝粒为基因胆道内组蛋白的系统操纵的新机会开辟了新的机会，以及对中心粒子层表层的全部表观遗传学特征的定义。带有条件性共粒的新型HAC还具有作为基因递送的系统的潜力，并在哺乳动物细胞中调节基因表达。为了使HAC中的调节基因表达可能，将含有HAC的TET-O从人类宿主细胞转移到表现出高度同源重组的鸡DT40细胞中。我们最近的研究表明，带有条件丝粒的HAC具有较高的有丝分裂稳定性，并在DT40细胞中的单个副本中保持。为了生成基于HAC的哺乳动物基因表达的系统，使用DT的重组机械（摘要以7800个字符的截断），将独特的LOX-P带到HAC中，并带有条件性的丝粒。

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