Comparative Analysis of Cancer-Associated Genes and Development of a Gene Delive

本研究的长期目标包括确定与前列腺癌易感性相关的Xq27染色体上的基因或基因组重排，并深入了解SPANX在正常和恶性细胞中的功能。一个潜在的影响深远的目标是确定人类细胞中新生着丝点形成的结构要求。成功实现这一目标将是朝着开发基于hac的人类疾病基因治疗和/或基因传递系统迈出的重要一步。1)遗传连锁研究提示Xq27位点的一个或多个基因与前列腺癌易感性有关。相应的区域横跨750 kb，包括5个SPANX基因（SPANX- a1, -A2, -B， -C和-D），它们编码在精核和多种癌细胞中表达的蛋白质。Xq27处的SPANX基因高度保守（即同源性为95%以上），并且位于大片段重复（SDs）中，这使得基于pcr的方法对这些基因进行突变分析变得复杂。然而，我们最近成功地对来自前列腺癌患者的xq27相关的SPANX基因进行了突变分析，使用转化相关重组（TAR）克隆并测序了这些基因。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，表明sd介导的涉及SPANX基因的同源重组可能导致遗传不稳定性增加，并可能提高SPANX基因的遗传多样性水平。突变分析结果显示，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。我们最近在人类基因组中发现了第二个SPANX基因家族，SPANX- n。spanxn基因簇包括spanxn - n1、spanxn - n2、spanxn - n3、spanxn - n4和spanxn - n5。spax - n蛋白彼此具有50-80%的同源性，与spax - a /D蛋白具有40-50%的同源性。鉴于SPANX基因在精子发生和癌症中的作用，我们将研究扩展到SPANX- n基因的进化、变异、表达调控和精子内定位。我们开发了一套spax特异性抗体，可以识别spax - a /D或spax - n蛋白。免疫荧光研究表明，spax - n蛋白和spax - a /D蛋白一样，只局限于减数分裂后的精细胞。然而，它们定位于顶体而不是核膜，并且在几种非性腺成人组织和许多癌细胞中表达水平较低。因此，我们的研究结果与最近SPANX基因的重复伴随着类人猿SPANX蛋白功能的多样化的可能性是一致的，这导致了spx - n和spx - a /D蛋白在减数分裂后精子中的差异定位以及spx - n蛋白在非性腺成年组织中的差异表达。对SPANX基因和基因产物的进一步研究可能会对精子发生和癌症发生以及基因组稳定性和多样化选择产生有价值的见解。2)利用携带合成阿尔法DNA阵列的HAC构建体分析了de novo着丝点形成的结构要求。我们的新RCA-TAR方法(7)利用酵母体内重组，从确定的低聚物底物中合成长度达120 kb的阿尔法DNA阵列。利用这些含阿尔法体DNA的构建体，我们正在寻求以下问题的答案：a)播种CENP-A染色质和稳定维持功能性着丝点所需的阿尔法体DNA的最小长度是多少；b) CENP-B结合位点在阿尔法DNA中的结构和/或功能作用是什么；c)向量序列在HAC构象中的作用。为了确定形成和维持一个功能性的人类着丝粒需要多少CENP-A染色质，构建了不同长度的α -卫星DNA阵列，并测试了它们成核CENP-A染色质和形成具有功能性着丝粒的HAC的能力。我们的研究结果表明，维持在30-70 kb阿尔法DNA阵列上的最小核心代表着着丝粒染色质的表观遗传记忆。一个可能的贡献的载体序列，呈现强制性的所有阿尔法DNA结构，对HAC的形成也进行了研究。我们最近的染色质免疫沉淀（ChIP）分析表明，虽然alphoid DNA促进CENP-A染色质和含有二甲基化组蛋白H3的染色质的组装，但载体衍生的Neo转录盒促进H3K4me2和H3K4me3染色质的组装（常染色质中的特征组蛋白修饰）以及H3K9me3染色质（异染色质的特征）。因此，载体衍生的转录盒可能对HACs的染色质结构和着丝粒功能有重要影响。利用RCA-TAR方法，我们构建了一种新的人类人工染色体（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。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。HAC为系统地操作着丝点内的组蛋白密码和定义着丝点染色质的完整表观遗传特征提供了新的机会。具有条件着丝粒的新型HAC在哺乳动物细胞中作为基因传递和调控基因表达的系统也具有很大的潜力。哺乳动物染色体的着丝粒区和近着丝粒区含有重复的DNA序列，显示出高速率的进化变化。这些序列在着丝点/异染色质结构和功能方面的确切作用尚不清楚。我们最近研究了几个重复元件对在小鼠红细胞白血病细胞中稳定整合到小鼠染色体中的转基因盒表达的影响。我们的研究结果表明，人类γ -卫星DNA来源于人类8号染色体的中心点周围区域，可以防止载体DNA诱导的eGFP报告基因的表观遗传沉默。电泳迁移位移分析显示，在人类染色体8、X和y的γ -卫星阵列中存在CTCF结合位点。我们还表明，这些位点与蛋白质结合独立于它们的甲基化状态。染色质免疫沉淀实验证实CTCF在体内结合这些位点。鉴于在大多数人类染色体中发现了γ -卫星DNA，这些数据表明，着丝粒周围γ -卫星DNA在维持人类着丝粒相关染色质的镶嵌结构中起作用，并可能阻止异染色质扩散到和/或超出着丝粒周围区域。γ -卫星DNA的强抗沉默活性也表明，这些序列可能有助于促进插入不同染色体位置和HAC的异位转基因的稳定表达

Long-term objectives of this research include identification of the gene(s) or genomic rearrangements on chromosome Xq27 that are involved in predisposition to prostate cancer, and insight into the functions of SPANX in normal and malignant cells. A potentially far-reaching objective is to define the structural requirements for de novo kinetochore formation in human cells. Success in achieving this goal would be an important step towards developing HAC-based gene therapy and/or gene delivery systems for human disease. 1) Genetic linkage studies implicate a gene or genes at Xq27 in prostate cancer susceptibility. 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. The SPANX genes at Xq27 are highly conserved (i.e., >95% homology) and reside within large segmental duplications (SDs), which complicates mutational analysis of these genes by PCR-based methods. However, we recently succeeded in performing mutational analysis of Xq27-linked SPANX genes from prostate cancer patients using transformation-associated recombination (TAR) to clone and sequence these genes. 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. The results of mutational 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 recently identified a second family of SPANX genes, SPANX-N, in the human genome. The SPANX-N gene cluster includes SPANX-N1, SPANX-N2, SPANX-N3, SPANX-N4 and SPANX-N5. SPANX-N proteins share 50-80% identity with each other and 40-50% identity with the SPANX-A/D proteins. Given the proposed role of SPANX genes in spermatogenesis and cancer, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. We developed a set of SPANX-specific antibodies that recognize SPANX-A/D or SPANX-N proteins. Immunofluorescence studies showed that SPANX-N proteins, like SPANX-A/D proteins, localize exclusively to post-meiotic spermatids. However they localize to the acrosome instead of the nuclear envelope, and they are expressed at a low level in several non-gonadal adult tissues as well as in many cancer cells. Thus, our findings are consistent with the possibility that the recent duplication of the SPANX genes was accompanied by diversification of the function of the SPANX proteins in hominoids, leading to differential localization of SPANX-N and SPANX-A/D proteins in post-meiotic sperm and differential expression of SPANX-N proteins in non-gonadal adult tissues. Additional study of SPANX genes and gene products is likely to yield valuable insight into spermatogenesis and carcinogenesis as well as genomic stability and diversifying selection. 2) The structural requirements for de novo kinetochore formation are being analyzed using HAC constructs carrying synthetic alphoid DNA arrays. Our novel RCA-TAR method (7) for constructing these arrays exploits in vivo recombination in yeast to generate synthetic alphoid DNA arrays up to 120 kb in length from defined oligomer substrates. Using these alphoid DNA-containing constructs, we are pursuing answers to the following questions: a) what is the minimal length of alphoid DNA required for the seeding of CENP-A chromatin and stable maintenance of a functional kinetochore; b) what is the structural and/or functional role of the CENP-B binding sites in alphoid DNA; and c) what is the role of vector sequences in a HAC formation. To determine how much CENP-A chromatin is required to form and maintain a functional human centromere, alpha-satellite DNA arrays in different lengths were constructed and tested on their ability to nucleate CENP-A chromatin and form a HAC with a functional kinetochore. Our results suggest that a minimum core maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin. A possible contribution of a vector sequence, that presents obligatory in all alphoid DNA constructs, on HAC formation was also investigated. Our recent chromatin immunoprecipitation (ChIP) analysis showed that while alphoid DNA promotes assembly of CENP-A chromatin and chromatin containing di-methylated histone H3, the vector-derived Neo transcriptional cassette promotes assembly of H3K4me2 and H3K4me3 chromatin (characteristic histone modifications in euchromatin) as well as H3K9me3 chromatin (characteristic of heterochromatin). Thus, the vector-derived transcriptional cassette may have a significant impact on chromatin structure and kinetochore function in HACs. Using the RCA-TAR method we have constructed a novel human artificial chromosome (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). 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. 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 has also a great potential as a system for gene delivery and regulated gene expression in mammalian cells. Centromeric and pericentromeric regions of mammalian chromosomes contain repetitive DNA sequences that exhibit a high rate of evolutionary changes. The exact role of these sequences with respect to kinetochore/heterochromatin structure and function is not understood yet. We recently investigated the effect of several repetitive elements on expression of a transgene cassette stably integrated into a mouse chromosome in mouse erythroleukemia cells. Our results show that human gamma-satellite DNA derived from the pericentromeric region of human chromosome 8 prevents epigenetic silencing of an eGFP reporter gene induced by a vector DNA. Electrophoretic mobility shift assay showed the presence of CTCF binding sites in the gamma-satellite arrays from human chromosomes 8, X and Y. We also showed that these sites are protein-bound independently of their methylation status. Chromatin immunoprecipitation experiments confirmed that CTCF binds these sites in vivo. Given the discovery of gamma-satellite DNA in most human chromosomes, these data suggest that pericentromeric gamma-satellite DNA plays a role in maintaining a mosaic structure in human centromeric-associated chromatin and may prevent heterochromatin spreading into and/or beyond the pericentromeric region. A strong anti-silencing activity of gamma-satellite DNA also suggests that these sequences may be useful to promote stable expression of ectopic transgenes inserted into different chromosomal locations as well as in a HAC