Human Artificial Chromosomes for Cancer Research and Functional Genomics

人类人工染色体（Human Artificial chromosome, HACs）是一种新型载体，在基因治疗、再生医学、抗癌药物筛选和生物技术等方面具有巨大潜力。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从细胞群中消除。通过在鸡DT40细胞中插入独特的基因受体loxP位点，tetO-HAC适于在人细胞中进行基因传递和基因表达。然后将修饰的HAC转移到仓鼠CHO细胞中，将感兴趣的基因插入HAC中，并通过微细胞介导的染色体转移（MMCT）将含基因的HAC转移到任何所需的受体细胞类型（Iida等，2010年）。随后证明了合成HAC在传递全尺寸基因和纠正人类细胞遗传缺陷方面的实用性。通过转化相关重组（TAR）克隆，分离出几种癌症相关基因的基因组拷贝，包括VHL和NBS1，并将其装载到HAC中，成功地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。在过去的一年里，我们主要对三个基因进行了分析。第一个基因PKD1编码多囊蛋白-1；该基因的突变可导致多囊肾病。第二个是TOP1mt，它编码线粒体拓扑异构酶i。TOP1mt基因之前在我们的分支中被发现。生化分析表明，MEF细胞中TOP1mt缺乏导致线粒体功能障碍，诱导DNA损伤反应（DDR）途径，并激活自噬。第三个基因是BRCA1。这些基因的基因组拷贝被装载到et- o HAC载体中，并转移到携带失活基因突变的细胞系中，以阐明基因的功能、调控及其与疾病的联系。最令人兴奋的结果是，HAC模块携带了90kb的BRCA1基因基因组拷贝。众所周知，BRCA1参与许多不同的细胞功能，然而，尚未发现将报道的BRCA1的生化活性与其肿瘤抑制功能联系起来的统一机制框架。我们首次证明，BRCA1缺陷导致高阶α -卫星重复序列（HORs）的转录特异性激活，这些重复序列组装在功能性着丝点的异染色质结构域中。同时，在组装到着丝染色质结构域的HORs中没有观察到可检测到的转录升高。众所周知，着丝点周围异染色质对着丝点的功能至关重要。因此，我们证明了BRCA1缺陷与着丝点功能障碍之间的联系。这支持了一种假设，即在BRCA1缺失的情况下，着丝点的表观遗传改变可能有助于细胞转化。与基于HAC的基因表达研究并行，我们继续优化et- o HAC载体本身。针对需要在HAC中插入多个基因的情况，设计并构建了多整合酶HAC载体。在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染色质结构域。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，我们比较了不同染色质绝缘体对装载到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。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。屏障元件，如γ -卫星DNA和tDNA（两者都是之前在我们的实验室中发现的），可以防止中染色质中的基因沉默，因此有助于优化使用HAC载体的转基因。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，我们描述了一种新的系统，该系统允许在没有转染步骤的情况下验证归因于HAC基因表达的表型变化。我们证明了携带4个由HAC组成表达的VP16结构域串联重复序列的单个tTA拷贝能够在HAC着丝粒中产生与其功能不相容的染色质变化。在新系统中，着丝细胞的失活是由强力霉素调控的。新改良的teto - haac系统在基因功能研究中具有多种应用潜力。我们还将tetO-HAC应用于影响染色体不稳定性（CIN）的药物筛选。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（Lee et al., 2013）。具体来说，在过去的一年里，我们比较了目前临床上使用的一系列不同的抑制剂，包括HDAC抑制剂、PARP抑制剂、微管稳定和微管不稳定药物、TOP1/TOP2抑制剂、Chk1/Chk2抑制剂、极光激酶抑制剂和核糖核苷酸还原酶抑制剂，它们诱导染色体丢失的能力。新发现的显著增加染色体错分离频率的化合物应该加速针对癌细胞CIN表型的新治疗策略的发展。在过去的一年里，我们努力使用半衰期缩短的改良EGFP（例如，带有degron box序列的EGFP）显著提高了基于hac的检测速度。我们的目标是将分析转化为高通量筛选。总之，tet-O HAC是表达全长基因和整个基因座以及纠正人类细胞遗传缺陷的最先进载体。同时，HAC提供了一个独特的机会来测量染色体不稳定性和筛选新的抗癌药物。我们新的基于hac的流式细胞术也可用于鉴定控制人类细胞中染色体分离的基因。此外，tetO-HAC也被用作研究表观遗传修饰在人类着丝点功能中的作用的独特系统（Bergmann et al., 2011, 2012）。最近，我们使用多整合酶HAC载体在现有的HAC中组装了一大块lacO/gal4阿尔法DNA阵列，以阐明异染色质在着丝点组装和维持中的作用。

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology. 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 eliminated from cell populations by inactivation of its conditional kinetochore. The tetO-HAC was adapted for gene delivery and gene expression in human cells by insertion of a unique gene acceptor loxP site in chicken DT40 cells. Then the modified HAC was transferred to hamster CHO cells where a gene of interest may be inserted into the HAC and from which the gene-containing HAC can be moved to any desired recipient cell type via microcell-mediated chromosome transfer (MMCT) (Iida et al 2010). It was subsequently 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 several cancer-associated genes, including VHL and NBS1, were isolated by transformation-associated recombination (TAR) cloning, loaded into the HAC and 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, we mainly focused on the analysis of three genes. The first gene, PKD1, encodes polycystin-1; mutations in this gene lead to polycystic kidney disease. The second is TOP1mt, which encodes a mitochondrial topoisomerase I. The TOP1mt gene has been previously discovered in our branch. Biochemical analyses indicate that TOP1mt deficiency in MEF cells results in mitochondrial dysfunctions, induction of the DNA damage response (DDR) pathways, and activation of autophagy. The third gene is BRCA1. Genomic copies of these genes were loaded into the tet-O HAC vectors and transferred into cell lines carrying inactivating mutations in the genes to clarify gene function, its regulation and link to disease. The most exciting results were obtained with the HAC module carrying the 90 kb genomic copy of the BRCA1 gene. As known, BRCA1 is involved in many disparate cellular functions, however, no unifying mechanistic framework that links the reported biochemical activity of BRCA1 to its tumor suppressor function has been identified yet. We demonstrated at first time that BRCA1 deficiency results in a specific activation of transcription of higher-order alpha-satellite repeats (HORs) assembled into heterochromatin domains in the functional kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. It is well known that of pericentromeric heterochromatin is essential for kinetochore function. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation. In parallel with HAC-based gene expression studies we continue to work on optimization of the tet-O HAC vector itself. For situations in which several genes need to be inserted into the HAC, a multi-integrase HAC vector was designed and constructed. In the tetO-HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While this domain is permissive for transcription, there are no studies on a long-term transgene expression within centrochromatin. In our recent study, we compared the effects of different chromatin insulators 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. 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 (both were previously discovered in our lab) 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 describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTA carrying 4 tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. In new system inactivation of kinetohore followed the HAC loss is regulated by doxycycline. The newly modified tetO-HAC-based system has the potential for multiple applications in gene function studies. We have also applied our tetO-HAC 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 (Lee et al., 2013b). Specifically, during the past year, a set of different inhibitors currently used in clinics, including HDAC inhibitors, PARP inhibitors, microtubule stabilizing and microtubule-destabilizing drugs, TOP1/TOP2 inhibitors, Chk1/Chk2 inhibitors, Aurora kinase inhibitors and ribonucleotide reductase inhibitors, were compared on their ability to induce chromosome loss. The new identified compounds that dramatically increase chromosome mis-segregation frequencies should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells. During the past year, we worked to significantly increase the speed of our HAC-based assay using a modified EGFP with a reduced half-life (e.g., EGFP with a degron box sequence). Our goal is to convert an assay into high-throughput screening. To summarize, the tet-O HAC is the most advanced vector for expression of full-length genes and entire loci and for correction of genetic deficiencies in human cells. At the same time, the HAC provides a unique opportunity to measure chromosome instability and screen new anticancer drugs. Our new HAC-based flow cytometry assay may be also applied for identifying genes controlling chromosome segregation in human cells. In addition, the tetO-HAC was also used as a unique system to study a role of epigenetic modifications in the human kinetochore function (Bergmann et al., 2011, 2012). Recently we used a multi-integrase HAC vector to assemble a large block of lacO/gal4 alphoid DNA arrays in the existing HAC to clarify the role of heterochromatin in kinetochore assembly and maintenance.