Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 8750677
• 负责人: Kyungjae Myung
• 金额: $ 75.26万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8750677
• 关键词: Affect; Aftercare; Aneuploidy; Aphidicolin; Ataxia Telangiectasia; BRCA1 Protein; BRCA2 gene; Biochemistry; Bypass; Cell Culture Techniques; Cell Death; Cell Nucleolus; Cell Proliferation; Cell Survival; Cells; Chromosomal Rearrangement; Chromosome Arm; Chromosomes; Collaborations; Complement; Complementary DNA; Complex; Congenital Abnormality; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA repair protein; DNA replication fork; Daphne plant; Development; Disease; Embryo; Endometrial Neoplasms; Fanconi anemia protein; Fanconi' s Anemia; Fungal Genome; Future; Gene Activation; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Goals; Hereditary Disease; Histone H3; Histones; Homologous Gene; Homologous Protein; Human; Human Cloning; Human Genome; Incidence; Inherited; Journals; Knockout Mice; Lead; Libraries; Link; Malignant Neoplasms; Mammals; Mediating; Metabolism; Methods; Modeling; Modification; Molecular; Molecular Biology; Mus; Mutate; Mutation; National Human Genome Research Institute; Open Reading Frames; Orthologous Gene; Pancytopenia; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Play; Postdoctoral Fellow; Predisposition; Prevention strategy; Proliferating Cell Nuclear Antigen; Proteins; Proto-Oncogenes; RNA Polymerase I; Regulation; Regulation of Proteolysis; Reporting; Ribosomal RNA; Role; S Phase; SMARCA3 gene; Sequence Homology; Signal Transduction; Sister Chromatid; Somatic Mutation; Source; Stress; Syndrome; System; Testing; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Work; Yeast Model System; Yeasts; Zebrafish; activator 1 protein; base; cancer cell; cancer therapy; genome database; genome wide association study; homologous recombination; human FRAP1 protein; human disease; hydroxyurea; interstitial; irradiation; malignant breast neoplasm; novel; null mutation; overexpression; prevent; protein complex; protein function; repaired; response; screening; small hairpin RNA; tumor; tumorigenesis; yeast genetics

Transmitting genetic information without creating deleterious genetic alterations is one of the most important tasks. Cells have evolved systems that check for and repair potentially lethal DNA damage. However, when these systems do not work properly, DNA damage accumulates and causes genetic changes or cell death. Accumulation of genetic changes, which is defined as a genomic instability is frequently observed in various types of genetic disorders including cancers. Genomic instability has been documented as a preceding step for multiple inactivations of tumor suppressor genes and activations of proto-oncogenes. One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangement (GCR). GCR includes translocations, deletions of chromosome arm, interstitial deletions, inversions, amplifications, chromosome end-to-end fusion and aneuploidy. Although little is known about the origin and mechanisms of GCRs observed in cancer cells, recent studies on genes mutated in inherited cancer predisposition syndromes have started to demonstrate that proteins that function in DNA damage responses, DNA repair, and DNA recombination, play crucial roles in the suppression of spontaneous and/or DNA damage-induced GCRs. The recent identification of strong correlations between genes responsible for genetic diseases including cancers and GCRs started to pinpoint the importance of GCRs. To understand mechanisms how GCRs are generated and how such GCR formation can lead tumorigenesis, we screened the entire yeast genome for mutations or overexpression that increase the rate of GCR formation. RAD5 and ELG1 from mutation screening and MPH1 from overexpression screening were selected for further studies of molecular mechanisms of these proteins to protect genome from deleterious GCR formation. 1. Determine the role of RAD5 orthologs in mammalian GCR and further dissect the RAD5 pathway upstream signals and additional factors. Previously, we identified two RAD5 orthologs in mammals and demonstrated that RAD5 orthologs, SHPRH and HLTF function to prevent collapse of persistent stalled replication forks by assisting template switching DNA damage bypass mechanism that uses the nascent strand of the sister chromatid for recombination mechanism for damage bypass. Among different modifications of Proliferating Cell Nuclear Antigen (PCNA) that determine the bypass mechanisms, we demonstrated that PCNA is poly-ubiquitinated by SHPRH and HLTF. Last year, we hypothesized that mice deficient in SHPRH would show a high incidence of tumorigenesis. We found that shprh-/- and hltf-/- and double knockout mice did not observe high level of tumorigenesis. In collaboration with Dr. Heinz Jacobs, we found that there is a redundant pathway that can complement the lack of SHPRH/HLTF pathway. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for SHPRH localization in the nucleolus. The localized SHPRH in the nucleolus regulate the transcription of rRNA and assist cellular proliferation. We found that rRNA expression by SHPRH was mediated by specific histone H3 modification and recruitment of RNA polymerase I. However, such new role of SHPRH in rRNA transcription was not affected by its original function that we found in DNA damage response. Lastly, we found that rRNA transcription by SHPRH depended on mTOR pathway that is important for cellular metabolism. 2. ATAD5 (mammalian ELG1 homolog): determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. To investigate whether the role of ELG1 in GCR suppression in yeast is conserved in mammals, we cloned the human ELG1 gene (ATAD5) by conducting a sequence homology search in the human genome database. Previously, we demonstrated that the reduced expression of the ATAD5 gene by shRNA increased spontaneous DNA damage resulted as evidenced by an increase of phosphorylated histone H2Ax and ATM foci. The ATAD5 protein was localized at the stalled replication fork after hydroxyurea treatment. We also demonstrated an increase of human ATAD5 expression at S-phase and after treatment of cells with various DNA-damaging agents, including MMS, hydroxyurea, aphidicolin, and gamma-irradiation. Previously, we demonstrated that ATAD5 interacts with PCNA and USP1 that removes ubiquitin from PCNA after DNA damage bypass pathway. In addition, we reported that mice haploinsufficient in Atad5 showed a high incidence of tumorigenesis. We recently confirmed that embryonic day 7.5 to 8.5 as embryonic lethality caused by homozygous null mutation of ATAD5. In addition, in collaboration with Dr. Daphne Bells group in NHGRI, we found human somatic mutations of ELG1 gene in many endometrial tumors. We also found several rare polymorphisms as well as cancer mutations in other tumor types. We are currently testing whether these rare mutations found in affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish. Lastly, in addition to its role in DNA repair, we found that ATAD5 functions in DNA replication through its interaction with PCNA. ATAD5 unloads PCNA when DNA replication ends at the end of S phase and at each cycle of lagging strand synthesis. 3. Determine the role of Mph1, the yeast homolog of FANCM, in DNA repair By screening genes that enhance GCR formation when overexpressed, we identified MPH1 as the strongest GCR enhancing gene. MPH1 has been implicated in a homologous recombination-dependent DNA repair pathway. Recently, the human homolog of MPH1 was discovered as the gene mutated in FA complementation group M (FANCM) patients. FA is a genomic instability disorder clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. The FA core complex consists of fourteen proteins participating in a DNA damage response network with breast cancer susceptible proteins, BRCA1 and BRCA2. Based on homology between MPH1 and FANCM, we hypothesized yeast also has a FA like pathway that functions for repair of intercrosslink (ICL) repair. ICLs covalently link complementary DNA strands, block DNA replication and transcription, and must be removed to allow cell survival. We genetically characterized a conserved yeast ICL repair pathway comprised of the yeast homologs (Mph1, Chl1, Mhf1, Mhf2) of four FA proteins (FANCM, FANCJ, MHF1, MHF2). We found that this pathway is epistatic with Rad5-mediated DNA damage bypass and distinct from the ICL repair pathways mediated by Rad18 and Pso2 that remove ICL damage by different mechanisms. In addition, consistent with FANCMs role in stabilizing ICL-stalled replication forks, we found that Mph1 prevents ICL-stalled replication forks from collapsing into double strand breaks. This unique repair function of Mph1 is specific for ICL damage and does not extend to other types of damage. These studies reveal the functional conservation of the FA pathway and validate the yeast model for future studies to further elucidate the mechanism of the FA pathway. Whole studies are reported in Journal of Biological Chemistry. Due to the departure of post-doctoral fellow who studied this project, the Mph1 project is now closed.

传输遗传信息而不创造有害的遗传改变是最重要的任务之一。细胞已经进化了系统，可以检查和修复潜在的致命DNA损伤。但是，当这些系统无法正常工作时，DNA损伤会积累并导致遗传变化或细胞死亡。在包括癌症在内的各种遗传疾病中经常观察到遗传变化的积累，被定义为基因组不稳定性。基因组不稳定性已被记录为肿瘤抑制基因的多种失活和原始癌基因激活的前一个步骤。在许多癌症中经常观察到的一种基因组不稳定性是染色体重排（GCR）。 GCR包括易位，染色体臂缺失，间质缺失，反转，放大，染色体端到端融合和非整倍性。尽管对在癌细胞中观察到的GCR的起源和机制知之甚少，但最近对遗传性癌症易感综合征突变的基因的研究开始证明，在DNA损伤反应，DNA修复和DNA重新组合中起作用的蛋白质在抑制自发性和/或DNA损害损害的GCR中起着至关重要的作用。最近鉴定了负责遗传疾病（包括癌症和GCR）的基因之间的牢固相关性，开始指出GCR的重要性。为了了解GCR的产生方式以及这种GCR形成如何导致肿瘤发生的机制，我们筛选了整个酵母基因组的突变或过表达，从而增加了GCR形成的速率。选择了从突变筛选和从过表达筛选中的MPH1进行的RAD5和ELG1，以进一步研究这些蛋白质的分子机制，以保护基因组免受有害GCR的形成。 1。确定RAD5直系同源物在哺乳动物GCR中的作用，并进一步剖析RAD5途径上游信号和其他因素。 以前，我们在哺乳动物中鉴定了两个RAD5直系同源物，并证明RAD5直系同源物，SHPRH和HLTF功能可以通过帮助模板开关DNA损伤机制来防止持续停滞的复制叉的崩溃，该模板使用姐妹染色剂的新生层损坏机制，以实现重组机制来实现重新组合机制来造成损坏。在确定旁路机制的增殖细胞核抗原（PCNA）的不同修饰中，我们证明了PCNA是由SHPRH和HLTF进行的聚泛素化。去年，我们假设缺乏SHPRH的小鼠会显示出较高的肿瘤发生率。我们发现SHPRH - / - 和HLTF - / - 以及双基因敲除小鼠没有观察到高水平的肿瘤发生。与Heinz Jacobs博士合作，我们发现有一条多余的途径可以补充SHPRH/HLTF途径的缺乏。 SHPRH具有一个独特的组蛋白相互作用域，称为PHD域。我们最近发现，该域对于核仁中的SHPRH定位很重要。核仁中的局部SHPRH调节rRNA的转录并有助于细胞增殖。我们发现，SHPRH的RRNA表达是由特异性组蛋白H3修饰和RNA聚合酶I的募集介导的。但是，SHPRH在rRNA转录中的新作用不受我们在DNA损伤反应中发现的原始功能的影响。 最后，我们发现SHPRH的rRNA转录取决于MTOR途径，这对于细胞代谢很重要。 2。ATAD5（哺乳动物ELG1同源物）：确定替代复制因子C（RFC）复杂蛋白是否指导DNA修复途径和复制。 为了研究ELG1在哺乳动物中的GCR抑制中的作用是否保守，我们通过在人类基因组数据库中进行序列同源性搜索来克隆人ELG1基因（ATAD5）。以前，我们证明，shRNA降低ATAD5基因的表达增加了自发性DNA损伤，从而证明了磷酸化的组蛋白H2AX和ATM焦点的增加。羟基脲处理后，ATAD5蛋白位于停滞的复制叉中。我们还证明了在S期和用各种DNA破坏剂（包括MMS，羟基脲，蚜虫和γ-辐照）处理细胞后，人ATAD5表达的增加。以前，我们证明了ATAD5与PCNA和USP1相互作用，这些PCNA和USP1在DNA损伤旁路途径后从PCNA中去除泛素。此外，我们报告说，ATAD5中的单倍弹性显示出较高的肿瘤发生率。我们最近证实，胚胎第7.5至8.5是由ATAD5的纯合无效突变引起的胚胎致死性。此外，与NHGRI的Daphne Bells Group合作，我们发现了许多子宫内膜肿瘤中ELG1基因的人类体细胞突变。我们还发现了其他肿瘤类型的几种罕见的多态性以及癌症突变。我们目前正在测试在ATAD5S分子功能中发现的这些罕见突变，并引起小鼠和斑马鱼中观察到的表型。最后，除了其在DNA修复中的作用外，我们还发现ATAD5通过与PCNA的相互作用而在DNA复制中起作用。 当DNA复制在S相结束时和滞后链合成的每个循环时，ATAD5卸载PCNA。 3。确定FANCM的酵母同源物MPH1在DNA修复中的作用 通过筛选过表达时增强GCR形成的基因，我们将MPH1确定​​为最强的GCR增强基因。 MPH1与同源重组依赖性DNA修复途径有关。最近，在FA互补组M（FANCM）患者中发现了MPH1的人类同源物。 FA是一种基因组不稳定性疾病，其临床特征是先天性异常，进行性骨髓衰竭和恶性肿瘤的倾向。 FA核心复合物由14种蛋白质组成，该蛋白与乳腺癌易感蛋白BRCA1和BRCA2一起参与DNA损伤反应网络。基于MPH1和FANCM之间的同源性，我们假设酵母还具有类似FA的途径，可用于修复间交叉修复（ICL）修复。 ICLS共价链接互补的DNA链，阻断DNA复制和转录，必须去除以允许细胞存活。我们在遗传上表征了由四种FA蛋白（FANCM，FANCM，FANCJ，MHF1，MHF2）组成的酵母同源物（MPH1，CHL1，MHF1，MHF2）组成的保守酵母ICL修复途径。我们发现，该途径是上皮的，具有RAD5介导的DNA损伤旁路，并且与由Rad18和PSO2介导的ICL修复途径不同，这些途径通过不同的机制消除了ICL损伤。此外，与幻想在稳定ICL恒星复制叉中的作用一致，我们发现MPH1防止ICL恒星的复制叉塌陷成双链断裂。 MPH1的这种独特的维修功能特异性对于ICL损坏，并且不会扩展到其他类型的损坏。这些研究揭示了FA途径的功能保护，并验证了酵母模型的未来研究，以进一步阐明FA途径的机制。整个研究在生物化学杂志上报道。由于研究了该项目的博士后研究员的离开，MPH1项目现已关闭。