Study of hereditary prostate cancer and human artificial chromosomes

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，导致该基因区域长时间重复DNA的广泛富集。由于其最近的扩增，在整个spax - a /D集群中，SPANX编码和侧翼序列几乎相同，这使得基于pcr的方法在寻找突变时对这些基因进行序列分析变得复杂。然而，我们最近成功地使用转化相关重组（TAR）技术对前列腺癌患者的xq27相关的SPANX基因进行了这样的分析，这使得直接从复杂的基因组中分离大的基因组片段成为可能。该分析揭示了Xq27处涉及SPANX基因的频繁基因缺失/重复和基于同源性的序列转移，这表明sd介导的涉及SPANX基因的同源重组可能导致种系中SPANX基因的遗传不稳定性增加，并可能提高遗传多样性水平。分析结果显示，SPANX基因簇的DNA序列变异和遗传单倍型与前列腺癌易感性无关。然而，xq27相关的前列腺癌易感性仍有可能与spax - a /D基因簇结构的变异有关。我们假设前列腺癌的x连锁易感性是由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，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/或表型改变和病理疾病。SPANX基因簇中的SDs密度异常高，占750 kb基因组区域的三分之一以上，这表明可能是基因组重排的热点。根据Xq27重复片段的结构和组织，预测SPANX基因和侧翼DNA的高缺失和重复以及含SPANX区域的反转。其中一些可能导致SPANX基因簇内或附近的一个或多个基因的表达改变。因此，未来的工作将集中在确定x连锁前列腺癌家族中可能导致恶性肿瘤的spax - a /D基因簇内的反转。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（即拷贝数为1）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/功能组织的机制；2)创造具有潜在治疗应用价值的基因传递载体提供了新的途径。直到最近，HACs是从现有的YAC或BAC文库中鉴定的50-100 kb的阿尔法体DNA片段构建的。通常，这些片段的完整DNA序列是未知的，这就不能对de novo着丝点形成的结构要求做出最终结论。在NCI建立我们的单位之后，我们将部分研究重点放在构建具有精确定义的DNA序列变异的合成阿尔法DNA阵列上。为此，我们开发了一种构建阿尔法DNA阵列的新方法，CADA，利用酵母体内重组。这种方法代表了一种突变分析阿尔法DNA的工具，因为每个构建的阵列都可以评估其形成HAC的能力，如果必要的话还可以进行修改。从阿尔法DNA底物中启动HAC形成的DNA序列组合的知识可能为哺乳动物着丝粒的功能组织提供独特的信息。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是：1)HAC形成的效率低；2)HAC形成时输入DNA的多聚性，使得基因拷贝数和基因在HAC中的位置难以控制。优化HAC结构的着丝粒成分可能是确保有效播种功能着丝粒的关键，也可能影响HAC形成过程中DNA多聚的程度。利用CADA方法，我们最近确定了能够形成HAC的阿尔法DNA阵列的最小尺寸，并构建了一个新的HAC来操纵活性着丝点内染色质的表观遗传状态。HAC具有二聚体α -卫星重复序列，包含一个具有CENP-B结合位点的天然单体和一个完全人工合成的单体，其CENP-B盒子被四环素操作符（tetO）取代。HAC表现出正常的着丝点蛋白组成和有丝分裂稳定性。将几种四环素抑制因子（tetR）融合到着丝粒中，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结构或使用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结合导致着丝粒丢失了CENP-A、CENP-B、CENP-C和H3K4me2，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的条件着丝粒为系统地操纵着丝粒内的组蛋白密码和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也具有在哺乳动物细胞中作为基因传递和调节基因表达系统的潜力。为了使HACs中的基因表达调控成为可能，将含有et- 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在DT40细胞中具有较高的有丝分裂稳定性，并维持在单个拷贝中。为了生成一个基于HAC的哺乳动物基因表达系统，使用DT的重组机制将一个独特的lox-P与条件着丝粒一起引入HAC[摘要剪切为7800个字符]

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]