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: 8175316
• 负责人: VLADIMIR LARIONOV
• 金额: $ 172.9万
• 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/8175316
• 关键词: Adopted; Affect; Aneuploidy; Antigens; Artificial Chromosomes; BRCA1 gene; Brothers; Cancer Patient; Cell Line; Cell Nucleus; Cells; Centromere; Chickens; Chimeric Proteins; Chinese Hamster Ovary Cell; Chromatin; Chromatin Modeling; Chromatin Structure; Chromosome Segregation; Chromosome Structures; Chromosome Transfer; Chromosome inversion; Chromosomes, Artificial, Human; Cloning; Code; Color; Complement; Complex; DNA; DNA Maintenance; DNA Sequence; DNA Sequence Rearrangement; Development; Disease; EP300 gene; Ectopic Expression; Epigenetic Process; Episome; Equilibrium; Evolution; Family Study; Future; Gene Cluster; Gene Deletion; Gene Delivery; Gene Duplication; Gene Expression; Generations; Genes; Genetic Transcription; Genetic Variation; Genome; Genomic Segment; Genomics; Goals; Hela Cells; Histones; Human; Human Cell Line; Human Genome; Individual; Inherited; Insertional Mutagenesis; Investigation; Kinetochores; Knowledge; Laboratories; Lead; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of testis; Mammalian Chromosomes; Maps; Mediating; Mitosis; Molecular; Mutagenesis; NBS1 gene; Nucleic Acid Sequence Homology; Patients; Pattern; Play; Polyploidy; Predisposition; Process; Proteins; Reporting; Research; Role; Satellite DNA; Source; Southern Blotting; Stem cells; Stretching; Structure; Susceptibility Gene; System; Tars; Techniques; Testing; Tetanus Helper Peptide; Tetracyclines; Therapeutic; Transcription Repressor/Corepressor; Transfection; Transferase; Transgenes; VHL gene; Work; base; c-Myc Staining Method; cancer cell; cell transformation; cell type; centromere protein A; chromosome loss; functional genomics; gene delivery system; gene therapy; genetic linkage; homologous recombination; human disease; in vivo; induced pluripotent stem cell; novel; novel strategies; physical mapping; programs; sperm cell; success; tumor progression; 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 region. Due to their recent amplification, both SPANX coding and flanking sequences in SDs are nearly identical throughout the SPANX-A/D cluster, which complicates mutational analysis of these genes by PCR. However, we succeeded in analysis of SPANX genes from prostate cancer patients, using TAR cloning 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 in this region might be a source for predisposition to hereditary prostate cancer. In our recent work, a search for large genomic rearrangements at Xq27 was undertaken using three-color FISH. This analysis was carried out with several extensively studied families with X-linked hereditary prostate cancer. Inversion of the chromosomal region including the SPANX gene cluster was found in affected but not in unaffected brothers. Thus our results are consistent with the hypothesis that this inversion is causally-related to susceptibility to prostate cancer in the affected patients. However, the molecular basis of such causal relationship is not known yet. We hypothesize that the inversion leads to activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. During the past year, we have concentrated on mapping the breakpoint(s) of the inversion. This analysis was performed using a combination of Southern blot-hybridization, PCR, TAR cloning and DNA sequencing. Several candidate breakpoint regions have been identified within 1 Mb genomic sequence corresponding to SPANX gene cluster and flanking sequences. Verification of these candidate regions is in progress. A future work will focus on physical mapping of breakpoint(s) and analysis how the inversion alters expression of genes at/near breakpoints 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 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) development of gene delivery vectors with potential therapeutic applications. The role of chromatin structure in kinetochore function has been studied intensively but still remains poorly understood. Recently we have generated a HAC in human HT1080 cells with a conditional centromere, which we expect to be instrumental in resolving many questions. The HAC includes approximately 6,000 copies of the tetracycline operator (tet-O) sequence. Such configuration allows a specific manipulation of the protein complement of a single kinetochore in vivo by targetting with tet-R fusion proteins. This approach has been used to target chromatin modifying proteins into the HAC and to demonstrate that a balance between open and condensed chromatin is critical for kinetochore function. The strongest effect on the synthetic kinetochore was observed after targeting of transcriptional repressors inducing HP1alpha-repressive chromatin. Our collaborative studies with William Earnshaws laboratory showed that the disruption of kinetochore structure by a transcriptional repressor reflects a hierarchical disassembly of kinetochore components reflecting a pattern of protein interactions within kinetochore. During the past year, we demonstrated that H3K4me2 chromatin domains are essential for maintaining centromere integrity. These results revealed a functional link between transcription potential of alphoid DNA and maintenance of the centrochromatin signature. This observation provides more evidence that centrochromatin resembles chromatin domains specific for actively transcribed genes. In future studies, our system will be used to investigate the structural/functional organization of the human kinetochore. Until recently, HACs were primarily generated and analyzed in HT1080 human cells. No HAC formation was observed in HeLa, BJ1 and TIG-7 cells. Our studies indicate that in these cells centromeric chromatin is highly enriched in H3K9me3 and HP1alpha compared to HT1080 cells. This suggests that differences in centromere heterochromatization may influence HAC formation and/or its stability in human cells. Indeed in our recent studies, we demonstrate that tethering of the histone trimethylase Suv39h1/KMT1A negatively regulates de novo CENP-A chromatin assembly and HAC formation on transfected alphoid DNAs carrying multiple tet-O sequences in HT1080, whereas tethering of histone acetyl-transferases p300/KAT3B or PCAF/KAT2B positively regulates HAC formation and kinetochore establishment in HeLa cells. These results indicate that chromatin assembly balance on alphoid DNA in the individual cell line is crucial for the de novo kinetochore assembly and that such an epigenetic barrier can be modulated. Practically, it means that de novo HAC formation may be induced in any type of human cells using alphoid DNA constructs with multiple tet-O sequences after their tethering by chromatin modifiers fused to the tet-repressor. HACs which carry a fully functional centromere are not associated with random mutagenesis and offer greater control over expression of ectopic genes. To adopt our HAC with a conditional centromere for gene delivery and gene expression in human cells, a loxP cassette was inserted into the HAC by homologous recombination in chicken DT40 cells following microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into chinese hamster ovary (CHO) cells, and a set of transgenes with the size up to 100 kb was efficiently and accurately incorporated into the tetO-HAC vector. The EGFP transgene (20 kb) was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted into the tet-O HAC can be eliminated from the cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy. Our future work will focus on expression of full-size human genes, VHL, NBS1, BRCA1, and ATM, in the HAC. After insertion of these genes into the HAC, it will be transferred into gene-deficient human cell lines via MMCT for complementation analysis. In addition, the tet-O-HAC will be tested for generation of iPS cells after insertion of the OCT4, SOX2, KLF4, cMYC cassette. Use of the HAC with a conditional centromere provides opportunity to eliminate the HAC along with the stem cell-inducing factors from the cell that allows to avoid insertional mutagenesis and cell transformation, complications that are frequently observed during cell re-programming.

遗传连锁研究表明Xq27位点的一个或多个基因与遗传性前列腺癌易感性（HPCX）有关。相应的区域横跨750 kb，包括5个SPANX基因（SPANX- a1, -A2, -B， -C和D），它们编码在精核和各种癌细胞中表达的蛋白质。每个SPANX基因都嵌入在一个最近形成的长达100 kb的片段重复（SD）中，导致该区域长段重复DNA的广泛富集。由于最近的扩增，SPANX编码和侧翼序列在整个SPANX- a /D集群中几乎相同，这使得PCR分析这些基因的突变变得复杂。然而，我们利用TAR克隆技术成功地分析了前列腺癌患者的SPANX基因，这使得从复杂的基因组中直接分离出大的基因组片段成为可能。该分析揭示了Xq27处SPANX基因频繁的基因缺失/重复和基于同源性的序列转移，提示sd介导的该区域同源重组可能是遗传性前列腺癌易感的一个来源。在我们最近的工作中，使用三色FISH进行了Xq27大基因组重排的搜索。这项分析是在几个被广泛研究的x连锁遗传性前列腺癌家族中进行的。包括SPANX基因簇在内的染色体区域倒置在受影响的兄弟中发现，但在未受影响的兄弟中没有发现。因此，我们的结果与假设一致，即这种倒置与受影响患者对前列腺癌的易感性有因果关系。然而，这种因果关系的分子基础尚不清楚。我们假设倒置导致SPANX蛋白表达的激活，该蛋白可能在多种人类细胞类型的癌症进展中发挥作用。在过去的一年里，我们专注于映射反演的断点。该分析采用Southern blot杂交、PCR、TAR克隆和DNA测序相结合的方法进行。在与SPANX基因簇和侧翼序列相对应的1 Mb基因组序列中发现了几个候选断点区域。对这些候选地区的核查正在进行中。未来的工作将集中在断点的物理定位和分析反转如何改变断点/附近可能导致恶性肿瘤的基因表达。经过二十多年的研究，人类着丝粒仍然是一个谜，人们对其知之甚少。Hunt Willards小组证明人类着丝粒中的原代DNA α卫星DNA （alphoid DNA）可以诱导人类HT1080细胞中着丝粒复合体的播种，这一领域取得了一些进展。几个研究小组已经证实了这一观察结果，并报道了人类人工染色体（HACs）在人类细胞中的形成，使用了涉及α卫星DNA的转染策略。这些HACs在细胞核中以单拷贝片段的形式存在，并具有完全功能的着丝点。HACs的发展和详细研究为：1)阐明新生着丝粒/着丝粒形成及其结构/功能组织机制；2)开发具有潜在治疗应用价值的基因传递载体提供了新的途径。染色质结构在着丝点功能中的作用已被深入研究，但仍知之甚少。最近，我们在人类HT1080细胞中产生了一个具有条件着丝粒的HAC，我们希望这对解决许多问题有帮助。HAC包括大约6000个四环素操作符（tet-O）序列。这样的结构允许通过靶向tet-R融合蛋白对体内单个着丝点的蛋白质补体进行特异性操作。这种方法已被用于将染色质修饰蛋白靶向到HAC中，并证明开放染色质和凝聚染色质之间的平衡对着丝点功能至关重要。对合成着丝点的影响最大的是靶向诱导hp1 -抑制染色质的转录抑制物。我们与William Earnshaws实验室的合作研究表明，转录抑制因子对着丝粒结构的破坏反映了着丝粒成分的分层分解，反映了着丝粒内蛋白质相互作用的模式。在过去的一年里，我们证明了H3K4me2染色质结构域对于维持着丝粒完整性至关重要。这些结果揭示了阿尔法DNA的转录潜能和着丝染色质特征的维持之间的功能联系。这一观察结果提供了更多的证据，表明着丝染色质类似于活跃转录基因特有的染色质结构域。在未来的研究中，我们的系统将用于研究人类着丝点的结构/功能组织。直到最近，HACs主要是在HT1080人类细胞中产生和分析的。HeLa、BJ1和TIG-7细胞未见HAC形成。我们的研究表明，在这些细胞中，与HT1080细胞相比，H3K9me3和HP1alpha的着丝粒染色质高度富集。这表明着丝粒异色化的差异可能影响人类细胞中HAC的形成和/或其稳定性。事实上，在我们最近的研究中，我们证明了组蛋白三甲基化酶Suv39h1/KMT1A的捆绑对HT1080中携带多个et- o序列的转染的脂蛋白dna上的从头CENP-A染色质组装和HAC的形成具有负调控作用，而组蛋白乙酰转移酶p300/KAT3B或PCAF/KAT2B的捆绑对HeLa细胞中HAC的形成和着丝点的建立具有正调控作用。这些结果表明，单个细胞系中阿尔法DNA上的染色质组装平衡对着丝点的重新组装至关重要，并且这种表观遗传屏障可以被调节。实际上，这意味着在染色质修饰剂与tet-阻遏因子融合后，在任何类型的人类细胞中，使用具有多个tet-O序列的alpha型DNA构建体，都可以诱导HAC的重新形成。携带功能齐全的着丝粒的HACs与随机突变无关，可以更好地控制异位基因的表达。为了将我们的HAC与条件着丝粒一起用于人细胞的基因传递和基因表达，我们在鸡DT40细胞微细胞介导的染色体转移（MMCT）后，通过同源重组将loxP盒插入HAC。然后将带有loxP盒式的tet-O HAC转移到中国仓鼠卵巢（CHO）细胞中，在tetO-HAC载体中高效、准确地植入了一组大小达100 kb的转基因。经MMCT转染后，EGFP基因在人细胞中稳定表达。由于插入到et- o HAC中的转基因可因着丝粒失活导致HAC丢失而从细胞中清除，因此该HAC载体系统提供了重要的新特性，并在基因表达研究和基因治疗方面具有潜在的应用前景。我们未来的工作将集中在全尺寸人类基因VHL、NBS1、BRCA1和ATM在HAC中的表达。将这些基因插入HAC后，通过MMCT将其转移到基因缺陷的人细胞系中进行互补分析。此外，在插入OCT4， SOX2, KLF4， cMYC卡带后，将测试tet-O-HAC是否产生iPS细胞。将HAC与条件着丝粒结合使用，可以消除细胞中的HAC和干细胞诱导因子，从而避免插入性突变和细胞转化，这是在细胞重编程过程中经常观察到的并发症。