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。在我们最近的工作中，使用三色间期FISH对SPANX区域的反转进行了搜索。这项分析是在几个广泛研究的Finish和JHU家族的x连锁遗传性前列腺癌患者和未受影响的对照组中进行的。在患病兄弟中检测到包括spax - b、spax - c、spax - a1、spax - a2和LDOC1基因区域的反转，而在未患病兄弟中未检测到。我们的结果与假设一致，即这种倒置与受影响患者的前列腺癌易感性有因果关系。然而，这种因果关系的分子基础尚不清楚。因此，未来的工作将集中于倒置的断点映射和分析倒置如何改变断点处/附近可能导致恶性肿瘤的基因表达。我们假设前列腺癌的x连锁易感性是由SPANX蛋白表达的激活引起的，该蛋白可能在多种人类细胞类型的癌症进展中发挥作用。由于其表达模式，SPANX基因是少数被认为是抗癌治疗潜在靶点的CT抗原之一。因此，我们启动了一个项目来评估SPANX抗原作为抗癌治疗药物的潜力。我们的研究表明，spax - b特异性T细胞前体存在于无癌个体中。此外，在体外免疫spxx - b处理的树突状细胞（dc）后，前体T细胞产生辅助性CD4+ T细胞和细胞溶解性CD8+ T细胞（即ctl）。重要的是，人类黑色素瘤细胞以非常高的水平表达spanxb，并在MHC I类分子上加工并呈递至少两个免疫优势的HLA-A2限制性spanxb表位，这些表位激活ctl介导的黑色素瘤细胞杀伤。综上所述，这些研究表明SPANX蛋白可能是抗癌治疗的新靶点，这可能与前列腺癌特别相关。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。然而，我们已经在人类HT1080细胞中产生了具有条件着丝粒的HAC，我们希望这有助于解决这个问题。这条HAC是芽殖酵母外的第一条染色体，具有受调节的着丝粒。HAC包括大约6000个四环素操作符（tet-O）序列。由于tet- o与tet抑制因子（tet- r）结合具有非常高的亲和力和特异性，因此该HAC中的tet- o序列阵列可以被tet- r融合蛋白有效靶向。该系统的强大之处在于，它允许在体内对单个着丝点的蛋白质补体进行特定的操作，同时使所有其他着丝点不受干扰。该系统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。在靶向转录抑制因子（tTS）诱导hp1α -抑制染色质后，观察到对合成着丝点的最强影响。我们最近与爱丁堡大学William Earnshaws实验室的合作研究表明，并非所有类型的抑制染色质都与着丝点功能不一致。因此，在着丝粒内建立PRC1 （Polycomb complex）抑制染色质对着丝粒的结构或功能没有影响。我们还表明，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。这些结果表明，这种新颖的着丝点解剖方法可能揭示着丝点内蛋白质相互作用的新模式。为了进一步研究该HAC的表观遗传修饰并使其用于全长人基因的表达，我们在鸡DT40细胞中通过同源重组将Lox-P - 5 HPRT - Hyg - TK盒插入HAC中。通过微细胞介导的染色体转移（microcell-mediated chromosome transfer， MMCT），将带有lox-P磁带盒的克隆转移回HPRT缺乏的仓鼠CHO细胞。结果表明，在CHO细胞中，20 kb的转基因基因可以高效、准确地插入改造后的ox- p HAC中，并且转基因基因稳定表达。由于CHO细胞响应秋碱形成微细胞的频率很高，因此HAC可以很容易地通过MMCT从供体CHO细胞转移到其他受体人或小鼠细胞系中。这种HAC系统的发展具有重要意义，因为它在基因治疗研究中具有潜在的未来应用前景。该系统也可用于生成多能干细胞和研究功能性人类着丝粒中的染色质动力学。α -卫星DNA是人类细胞中唯一具有已知功能的着丝粒DNA，它能促进着丝粒的形成。在人类着丝粒中也发现了非阿尔法DNA重复序列。然而，人类染色体中非阿尔法染色体邻近中心重复DNA的精确序列和顺序仍未确定。由于异染色质的扩散是一个活跃的过程，人类着丝粒的多结构域组织表明存在特定的元件来划分不同的染色质状态。到目前为止，很少有被称为染色质屏障的具有这种活性的元素被描述。在我们最近的工作中，我们已经表明，所有人类染色体的中心点周围区域都含有伽马卫星DNA。使用一个新的实验系统，我们证明了人类近中心点γ卫星DNA作为异染色质阻滞元件的功能。在造血细胞中，γ -卫星DNA的抗沉默和异染色质阻滞活性需要与Ikaros结合，Ikaros是一种调节造血的蛋白质。γ -卫星DNA阵列在非造血细胞（即Ikaros不表达的细胞）中促进开放染色质结构的能力表明，在其他细胞类型中，另一种蛋白质可能与这些重复序列结合。我们提出，人类γ -卫星DNA的主要作用可能是防止中心周围异染色质向染色体扩散[摘要截断为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. In our recent work, a search for inversions in the SPANX region was undertaken using three-color interphase FISH. This analysis was performed with prostate cancer patients and unaffected controls from several extensively studied Finish and JHU families with X-linked hereditary prostate cancer. An inversion in the region including SPANX-B, SPANX-C, SPANX-A1, SPANX-A2, and the LDOC1 gene was detected in affected but not in unaffected brothers. 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 a causal relationship is not yet known. Therefore, future work will focus on mapping of breakpoint(s) of the inversion and on the analysis how the inversion alters expression of genes at/near breakpoints which could lead to malignancy. We hypothesize that X-linked predisposition to prostate cancer is caused by activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. Because of their expression pattern, SPANX genes are among the few CT antigens that are considered to be potential targets for anticancer therapeutics. Thus, we initiated a project to assess the potential of SPANX antigens as anticancer therapeutics. Our studies demonstrated that SPANX-B-specific T cell precursors are present in cancer-free individuals. Furthermore, after in vitro immunization with SPANX-B -treated dendritic cells (DCs), the precursor T cells give rise to helper CD4+ T cells and cytolytic CD8+ T cells (i.e. CTLs). Importantly, human melanoma cells express SPANX-B at a very high level and process and present at least two immunodominant HLA-A2 restricted epitopes of SPANX-B on MHC class I molecules, which activate CTLmediated killing of melanoma cells. Taken together, these studies indicated that SPANX proteins could be novel targets of anticancer therapeutics, which may be particularly relevant to prostate cancer. The role of chromatin structure in kinetochore function has been studied intensively but remains poorly understood. However we have generated a HAC in human HT1080 cells with a conditional centromere, which we expect to be instrumental in resolving this question. This HAC is the first chromosome outside of budding yeast with a regulated centromere. The HAC includes approximately 6,000 copies of the tetracycline operator (tet-O) sequence. Because tet-O is bound with very high affinity and specificity by the tet repressor (tet-R), the array of tet-O sequences in this HAC can be targeted efficiently with tet-R fusion proteins. The power of this system is that it allows the specific manipulation of the protein complement of a single kinetochore in vivo, while leaving all other kinetochores unperturbed. This system 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 a transcriptional repressor (the tTS) inducing HP1alpha-repressive chromatin. Our recent collaborative studies with William Earnshaws laboratory at Edinburgh University showed that not all types of repressive chromatin are inconsistent with kinetochore function. Thus, establishment of PRC1 (Polycomb complex) repressive chromatin within the centromere has no effect on kinetochore structure or function. We also have shown that the disruption of kinetochore structure by a transcriptional repressor reflects a hierarchical disassembly of kinetochore components. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore. To extend studies of epigenetic modification of this HAC and to adopt it for expression of full length human genes, a Lox-P - 5 HPRT - Hyg - TK cassette was inserted into the HAC by homologous recombination in chicken DT40 cells. Clones with the lox-P cassette were transferred back into hamster CHO cells deficient in HPRT via microcell-mediated chromosome transfer (MMCT). We demonstrated that 20 kb transgenes can be efficiently and accurately inserted into the retrofitted lox-P HAC in CHO cells and the transgenes are stably expressed. Because CHO cells form microcells at high frequency in response to colcemid, the HAC can be easily moved from donor CHO cells into other recipient human or mouse cell lines via MMCT. The development of this HAC system is important, because of its potential future use in gene therapy research. This system may also be useful to generate pluripotent stem cells and for studies of chromatin dynamics in a functional human centromere. Alpha-satellite DNA, which can promote kinetochore formation in human cells, is the only human centromeric DNA with a known function. Non-alphoid DNA repeats have also been identified in human centromeres. However, the precise sequence and order of non-alphoid pericentromeric repetitive DNA in human chromosomes remains mostly undetermined. Because the spreading of heterochromatin is an active process, the multi-domain organization of the human centromere suggests the presence of specific elements demarcating the different chromatin states. So far, very few elements with such activity, known as chromatin barriers, have been described. In our recent work, we have shown that pericentromeric regions of all human chromosomes contain gamma-satellite DNA. Using a new experimental system, we demonstrated that human pericentromeric gamma-satellite DNA functions as a heterochromatin arresting element. In hematopoietic cells, the anti-silencing and heterochromatin-arresting activities of gamma-satellite DNA require the binding of Ikaros, a protein that regulates hematopoeiesis. The ability of gamma-satellite DNA arrays to promote open chromatin structures in non-hematopoietic cells (i.e. where Ikaros is not expressed) suggests that another protein(s) may bind to these repeats in other cell types. We propose that the primary role of human gamma-satellite DNA may be to prevent the spreading of pericentric heterochromatin into chromosoma [summary truncated at 7800 characters]