Study of hereditary prostate cancer and human artificial chromosomes

一些连锁研究提供了在Xq27-q28处存在遗传性前列腺癌位点HPCX1的证据。然而，该地区的易感基因尚未确定。前列腺癌最强的连锁峰位于Xq27上750-kb的可变区域，该区域富含携带一簇SPANX基因的大SDs。SPANX基因未检测到突变。提示前列腺癌的易感性可能是由SDs之间的重组相互作用引起的一种基因组疾病。自上次年度报告以来，我们集中分析了几个与HPCX1联系最强的x连锁家族的750-kb区域。通过酵母体内重组（一种TAR克隆技术）直接分离出一组重叠的基因组片段，这些片段携带的SDs完全覆盖了750-kb区域，并对该区域进行了突变分析。对分离片段的后续分析排除了Xq27上750-kb的遗传不稳定区域作为前列腺恶性肿瘤的候选位点。当我们的工作完成并提交发表时（基因，染色体与癌症，2012,51:933-948），国际前列腺癌遗传学协会重新评估了先前的连锁数据，并得出结论，没有强有力的证据表明位于Xq27-28的主要前列腺癌易感基因（Bailey-Wilson JE et al；国际前列腺癌遗传学协会）。国际前列腺癌遗传联盟（ICPCG）家族Xq27-28连锁分析[j] .中华医学杂志，2012,13:46。在排除了Xq27上750-kb的遗传不稳定区域作为前列腺恶性肿瘤的候选位点后，我们的实验室已经完成了与该子项目相关的研究，我们将重点放在与人类人工染色体（Human Artificial chromosome, HACs）相关的其他子项目上。由阿尔法DNA阵列组装而成的HACs为功能基因组学和基因治疗提供了一种新的表观体基因传递载体。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。我们之前构建了一种合成的HAC (tetO-HAC)，它可以通过使其条件着丝点失活而很容易地从细胞群中消除。这种HAC是最先进的全长基因和整个基因座的表达载体，也是人类细胞遗传缺陷的校正载体。tetO-HAC也被用作一个独特的系统来研究表观遗传修饰在人类着丝点功能中的作用。tetO-HAC的广泛应用需要对其结构组织有一定的了解。在过去的一年里，我们完成了在HAC中由合成teto阵列形成的超大碱基大小的合成阿尔法DNA阵列的物理表征。我们的研究结果为在基因装载和HAC转移到不同宿主细胞过程中控制tetO-HAC的结构完整性提供了一种工具。它们还揭示了人类细胞中HAC从头形成的机制。我们之前证明了合成HAC在人类细胞中用于传递全尺寸基因和纠正遗传缺陷的效用。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，当细胞通过使增殖细胞群体中的着丝点失活而治愈HAC时，由HAC稳定基因表达引起的表型可以逆转。在过去的一年中，其他几个人类基因被加载到HAC中进行基因转移/基因表达研究，包括BRCA1和mtTOP1基因，这些基因之前在LMP中发现。在有条件着丝粒的tetO-HAC中，一个基因装载位点被插入对着丝粒组装和维持至关重要的着丝粒结构域。虽然该结构域允许转录，但没有关于着丝染色质内长期转基因表达的研究。在我们最近的研究中，我们比较了三种染色质绝缘体cHS4， γ -卫星DNA和tDNA对装载到tetO-HAC载体上的EGFP转基因表达的影响。出乎意料的是，绝缘子的功能对于转基因在染色质中稳定表达是必不可少的，而染色质是开放的。由两个tRNA基因的功能拷贝组成的tDNA绝缘体（在我们的实验室中发现）显示出最高的屏障活性。我们推断，靠近着染色质并不能保护缺乏染色质绝缘子的基因免受表观遗传沉默。阻止中染色质中基因沉默的屏障元件，如γ -卫星DNA和tDNA，将有助于利用HAC载体优化转基因。与其他HAC载体相比，tetO-HAC具有优势，因为它可以通过染色质修饰剂（如tTS）与着丝粒tetO序列结合，使HAC着丝粒失活，从而很容易从细胞中消除。诱导HAC丢失的机会为加载到α -HAC的基因诱导的表型提供了独特的控制。然而，HAC着丝点的失活需要逆转录病毒载体转染细胞以实现高水平的tTS表达，这一步骤可能导致插入性突变。在我们最近的工作中，我们描述了一种重新设计alphoidtetO-HAC载体的方法，该方法允许在不转染外源染色质调节剂的情况下验证归因于HAC基因表达的表型变化。在新的HAC载体中，将tTS盒与感兴趣的基因一起插入基因装载位点。在缺乏强力霉素的情况下，tTS的表达在α -HAC上产生自我调节的波动异染色质，诱导HAC上的基因快速而强烈的沉默，而对HAC的分离没有显著影响。这种沉默是可逆的，因此通过添加强力西环素可以很容易地恢复hac编码基因的表达。新修饰的基于alphoidteto - hac的系统在基因功能研究中具有多种应用潜力。我们还应用我们的HAC系统筛选影响染色体不稳定性（CIN）的药物。虽然CIN可以作为癌症基因组进化和肿瘤进展的驱动因素，但最近的研究结果表明，存在一个阈值水平，超过这个阈值，CIN就会成为肿瘤生长的障碍。我们的目标是开发一种新的定量分析方法来鉴定提高癌细胞中CIN的药物。为此，使用Cre-loxP重组将EGFP转基因加载到tetO-HAC中。EGFP的存在允许用流式细胞术测量HAC的损失。我们已经成功地使用这种方法来测量不同抗癌药物对染色体错误分离的影响。增加染色体错分离率的新化合物的鉴定应该加速针对癌细胞CIN表型的新治疗策略的发展。

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. However, the susceptibility gene in this region has not yet been identified. The strongest linkage peak of prostate cancer overlies a variable region of 750-kb at Xq27 that is enriched by large SDs carrying a cluster of SPANX genes. No mutations were detected in SPANX genes. It suggests that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. Since the last Annual Report, we have concentrated on the analysis of the 750-kb region in several X-linked families with the strongest linkage to HPCX1. Direct isolation of a set of overlapping genomic segments carrying SDs that completely covers the 750-kb region by in vivo recombination in yeast (a TAR cloning technique) were used to perform a mutational analysis of this region. The subsequent analysis of isolated fragments excluded the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. When our work was completed and submitted for publication (Genes, Chromosomes & Cancer J 2012, 51:933-948), the International Consortium for Prostate Cancer Genetics re-evaluated previous linkage data and concluded that there is no strong evidence for a major prostate cancer susceptibility gene located at Xq27-28 (Bailey-Wilson JE et al; International Consortium for Prostate Cancer Genetics. Analysis of Xq27-28 linkage in the international consortium for prostate cancer genetics (ICPCG) families BMC Med Genet 2012, 13: 46.) After exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy, the studies related to this sub-project have been completed in our lab and we have focused on other sub-projects linked with Human Artificial Chromosomes (HACs). (HACs) assembled from alphoid DNA arrays represent a novel episomal gene delivery vector for functional genomics and gene therapy. 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 constructed a synthetic HAC (tetO-HAC) that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This HAC is the most advanced vector for expression of full-length genes and entire loci and for correction of genetic deficiencies in human cells. The tetO-HAC was also used as a unique system to study a role of epigenetic modifications in the human kinetochore function. The broad use of the tetO-HAC requires the knowledge of its structural organization. During the past year, we completed physical characterization of a megabase- size synthetic alphoid DNA array in the HAC that has been formed from a synthetic tetO-array. Our results provide a tool to control structural integrity of tetO-HAC during gene loading and HAC transfer into different host cells. They also shed light on a mechanism for de novo HAC formation in human cells. We previously demonstrated 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 have also shown 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. During the past year, several other human genes were loaded into the HAC for gene transfer/gene expression studies, including BRCA1 and mtTOP1 gene previously discovered in LMP. In the tetO-HAC with a conditional centromere, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While this domain is permissivefor transcription, there are no studies on a long-term transgene expression within centrochromatin. In our recent study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA and tDNA, on the expression of an EGFP transgene loaded into the tetO-HAC vector. Unexpectedly, insulator function was essential for stable expression of the transgene in centrochromatin that represents open chromatin structure. A tDNA insulator (discovered in our lab) consisting of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements, such as gamma-satellite DNA and tDNA, that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the alphoidtetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector to achieve a high level of tTS expression, a step that potentially may lead to insertional mutagenesis. In our recent work, we described an approach to re-engineering the alphoidtetO-HAC vector that allows verification of phenotypic changes attributed to expression of genes from the HAC without transfecting exogenous chromatin modulators. In the new HAC vector, a tTS cassette is inserted into a gene-loading site along with a gene of interest. In the absence of doxycycline, expression of the tTS generates a self-regulating fluctuating heterochromatin on the alphoidtetO-HAC that induces a fast and strong silencing of the genes on the HAC without significant effect on HAC segregation. This silencing is reversible, and therefore expression of the HAC-encoded genes can be readily recovered by adding doxycycline. The newly modified alphoidtetO-HAC-based system has the potential for multiple applications in gene function studies. We have also applied our HAC system for screening of drugs affecting chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth. Our goal was to develop a new quantitative assay for identification of drugs that elevate CIN in cancer cells. For this purpose, the EGFP transgene was loaded into the tetO-HAC using Cre-loxP recombination. The presence of EGFP allows measuring of the HAC loss by flow cytometry. We have successfully used this assay to measure increased mis-segregation of chromosomes in response to different anticancer drugs. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.