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，这些基因编码在精子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。我们之前的分析显示，在Xq27位点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。为了支持这一假设，三色FISH在该区域检测到较大的反转。在过去的一年中，我们专注于绘制前列腺癌患者反转的断点。在与SPANX基因簇对应的750 kb序列中发现了几个候选断点区域。采用Southern blot杂交、long PCR、TAR克隆和DNA测序相结合的方法进行分析。对这些候选地区的核查正在进行中。我们假设这种类型的重排导致SPANX蛋白表达的激活，这可能在多种人类细胞类型的癌症进展中发挥作用。人类人工染色体（Human Artificial chromosome, 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。三年前，我们已经在人类HT1080细胞中产生了一个具有条件着丝粒的HAC，我们希望这将有助于解决许多问题。HAC包括大约6000个四环素操作符（tetO）序列拷贝。这种结构允许通过靶向tetR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。这种方法以前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。对合成着丝点影响最大的是系缚转录抑制物诱导的hp1 α抑制染色质。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，我们证明了在着丝粒相关的染色质中捆绑一个最小的NFKB p65激活域会产生具有高水平H3K9ac和转录物水平10倍升高的染色质，但对着丝粒组装或功能没有显著影响。相比之下，捆绑疱疹病毒VP16激活域产生了类似的染色质变化，但导致转录物的150倍升高，接近内源性内源家政基因的转录水平。这迅速使着丝点失活，导致组装的CENPA丢失并阻止进一步的CENPA组装。我们的数据揭示了体内功能着丝粒的显著可塑性：着丝粒可以忍受染色质环境的深刻变化，但似乎对着丝粒转录水平非常敏感。此外，我们发现人类着丝体DNA阵列上的新CENPA组装和着丝点形成受组蛋白H3K9乙酰/甲基开关的调节。将组蛋白乙酰转移酶（HATs）拴在阿尔法体DNA阵列上，打破了一个细胞类型特异性屏障，使CENPA重新稳定组装，并诱导其他着丝点蛋白在异位阿尔法体位点组装。将CENPA沉积因子hMis18a或HJURP系在alphoid阵列上后，也观察到类似的效果。相反，将H3K9三甲基化酶（Suv39h1）拴在阵列上会导致H3K9甲基化，从而阻止从头组装CENPA和着丝点形成。HAT系接绕过了对hMis18a的需要，但不需要HJURP来在异位位点重新组装CENPA、CENPT、CENPI和CENPE。通过这种机制重新组装的CENPA阵列可以形成人类人工染色体（HACs）的着丝点，并在人类细胞中无限繁殖。基于HAC的载体为任何大小的全长人类基因的传递和表达提供了一个有前途的系统。HACs避免了有限的克隆能力，缺乏拷贝数控制和插入突变，由于整合到宿主染色体困扰病毒载体。我们之前在合成的tetO HAC中引入了一个独特的基因受体位点，通过使其条件着丝点失活，可以很容易地从细胞群体中消除。在我们最近的工作中，我们展示了合成HAC在人类细胞中传递全尺寸基因和纠正遗传缺陷方面的效用。研究人员成功地将von Hippel Lindau综合征（VHL）中突变的VHL和Nijmegen breaking syndrome （NBS）中突变的NBS1两种癌症相关基因的基因组拷贝转移到基因缺陷细胞中。我们还表明，当细胞在增殖细胞群中通过使HAC的着丝点失活来治愈HAC时，由HAC稳定基因表达引起的表型可以逆转。酵母中Pol III转录的tRNA基因具有染色质屏障元件的功能，这是一个公认的事实。然而，到目前为止，还没有实验证据表明tRNA和其他Pol III转录基因在哺乳动物中表现出屏障活性。我们最近的结果为小鼠基因组中的类似现象提供了证据，小鼠基因组中含有大约1000倍于酵母基因组的假定RNA Pol III转录基因。因此，我们的研究结果表明，tRNA基因是哺乳动物细胞中染色质结构域结构建立和维持的基本要素。合成tRNA基因衍生的屏障元件以及其他已知的屏障元件现在正被纳入基于（HAC）的具有条件着丝粒的基因传递系统中。最终，我们期望开发一种基于HAC的系统，实现全尺寸人类基因的一致调控表达，将Pol II依赖性转录单位不受控制的表观遗传沉默风险降至最低。

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, 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. Our previous 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 support of this hypothesis, a large inversion was detected in this region by three color FISH. During the past year, we have concentrated on mapping the breakpoint(s) of an inversion in prostate cancer patients. Several candidate breakpoint regions have been identified within a 750 kb sequence corresponding to the SPANX gene cluster. This analysis was performed using a combination of Southern blot-hybridization, long PCR, TAR cloning and DNA sequencing. Verification of these candidate regions is in progress. We hypothesize that such type of rearrangements leads to activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. The development and detailed studies of Human Artificial Chromosomes (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. Three years ago 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 (tetO) sequence. Such configuration allows a specific manipulation of the protein complement of a single kinetochore in vivo by targetting with tetR fusion proteins. This approach has been previously 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 tethering 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 tethering a minimal NFKB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of H3K9ac and a 10 fold elevation in transcript levels, but had no significant effect on kinetochore assembly or function. In contrast, tethering the herpes virus VP16 activation domain produced similar chromatin changes but resulted in a 150 fold elevation in transcripts approaching the level of transcription at an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing loss of assembled CENPA and blocking further CENPA assembly. Our data uncover a remarkable plasticity of functional centromeres in vivo: kinetochores tolerate profound changes in their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription. In addition, we discovered that de novo CENPA assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl switch. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENPA assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar effects were observed after tethering of CENPA deposition factors hMis18a or HJURP, to the alphoid array. In contrast, tethering of H3K9 tri-methylase (Suv39h1) to the array causes methylation of H3K9, preventing de novo CENPA assembly and kinetochore formation. HAT tethering bypasses the need for hMis18a, but not HJURP for de novo CENPA, CENPT, CENPI, and CENPE assembly at the ectopic site. CENPA arrays assembled de novo by this mechanism can form kinetochores of human artificial chromosomes (HACs) that are propagated indefinitely in human cells. HAC based vectors offer a promising system for delivery and expression of full length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. We previously introduced a unique gene acceptor site into the synthetic tetO HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. In our recent work, we demonstrate the utility of the synthetic HAC for delivery of full size genes and correction of genetic deficiencies in human cells. Specifically genomic copies of two cancer-associated genes, VHL mutated in von Hippel Lindau syndrome (VHL) and NBS1 mutated in Nijmegen breakage syndrome (NBS) were successfully transferred into gene deficient cells. We also show that phenotypes arising from stable gene expression from the HAC can be reversed when cells are cured of the HAC by inactivating its kinetochore in proliferating cell populations. It is a well established fact that the Pol III transcribed tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III transcribed genes exhibit barrier activity in mammals. Our recent results present evidence for a similar phenomenon in the mouse genome, which contains approximately 1000-times more putative RNA Pol III transcribed genes than the yeast genome. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells. Synthetic tRNA genes derived barrier elements as well as other known barrier elements are now being incorporated into a (HAC) based gene delivery system with a conditional centromere. Ultimately, we expect to develop a HAC based system that achieves consistent regulated expression of full size human genes, with minimal risk of uncontrolled epigenetic silencing of Pol II dependent transcription units.