Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 8948363
• 负责人: Kyungjae Myung
• 金额: $ 68.42万
• 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/8948363
• 关键词: Affect; Alleles; Aneuploidy; Bypass; Cell Culture Techniques; Cell Death; Cell Nucleolus; Cells; Chromosomal Rearrangement; Chromosome Arm; Chromosomes; Collaborations; Core Facility; DNA Damage; DNA Repair; DNA Repair Pathway; DNA repair protein; DNA replication fork; DNA-dependent protein kinase; Daphne plant; Databases; Development; Ectopic Expression; Embryo; Endometrial Neoplasms; Fertilization; Gene Activation; Gene Silencing; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Goals; Hereditary Disease; Histones; Homologous Gene; Human; Incidence; Inherited; International; Knockout Mice; Lead; Malignant Neoplasms; Mammals; Modeling; Modification; Molecular; Mus; Mutate; Mutation; National Human Genome Research Institute; Orthologous Gene; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Play; Predisposition; Prevention strategy; Proliferating Cell Nuclear Antigen; Proteins; Proto-Oncogenes; Regulation; Reporting; Ribosomal RNA; Role; SMARCA3 gene; Serine; Sister Chromatid; Site-Directed Mutagenesis; Somatic Mutation; Source; Syndrome; System; Testing; The Cancer Genome Atlas; Tumor Suppressor Proteins; Ubiquitination; Variant; Work; Zebrafish; activator 1 protein; cancer cell; cancer genome; cancer therapy; carcinogenesis; hatching; human FRAP1 protein; human disease; in vivo; interstitial; novel; null mutation; overexpression; prevent; protein complex; protein function; repaired; response; screening; tissue culture; tumor; tumorigenesis; yeast genome; zygote

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. 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 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, SHPRH. 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. Although we tested for association of SHPRH and HLTF with tumorigenesis in vivo, the inactivation of these genes did not result in an increase in tumorigenesis. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for SHPRH localization in the nucleolus. In our characterization of the general DNA damage response regulating PCNA ubiquitination, we unexpectedly found that phosphorylation of Serine 4 and 8 of RPA32, which we used as a DNA damage response control, depends on DNA dependent protein kinase. We found a novel function of SHPRH in the nucleolus that drove the project into a novel molecular mechanism of SHPRH in rRNA transcription in the nucleolus. We found the function of SHPRH for rRNA transcription is mTOR-dependent. 2. ATAD5 (mammalian ELG1 homolog): determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. 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 ATAD5 gene in many endometrial tumors. We also found several rare polymorphisms as well as cancer mutations in other tumor types. We recovered a zebrafish atad5 null allele from the TILLING project in the NHGRI zebrafish core facility. Unlike the atad5 null mouse, which dies at embryonic day 8.5, an atad5 null zebrafish survives until seven days post fertilization (dpf). The lethal phenotype of the atad5 null zebrafish became obvious right after hatching when fertilized eggs were treated with MMS. Therefore, we believe that in vivo functional activity of ATAD5 variants identified can be tested with the atad5 null zebrafish model. We have generated various ectopic expression constructs with a single sequence variant found in somatic cancer mutations as well as database search in the International Cancer Genome Consortium for the Cancer Genome Atlas using site-directed mutagenesis. We are currently testing whether these rare mutations found in affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish as well as in tissue culture system.

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。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。从突变筛选中选择了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直系同源物的作用，shprh。 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。 尽管我们测试了SHPRH和HLTF与体内肿瘤发生的缔合，但这些基因的失活并未导致肿瘤发生增加。 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。在调节PCNA泛素化的一般DNA损伤反应的表征时，我们出乎意料地发现，RPA32的丝氨酸4和8的磷酸化（我们用作DNA损伤响应控制）取决于DNA依赖性蛋白激酶。我们在核仁中发现了SHPRH的新功能，该功能将项目推向了核仁中rRNA转录中SHPRH的新分子机制。我们发现SHPRH对于rRNA转录的功能是mTOR依赖性的。 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。 我们报道说，ATAD5中富含的小鼠表现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ATAD5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。我们从NHGRI斑马鱼核心设施的耕作项目中收回了斑马鱼ATAD5 NULL等位基因。与在胚胎第8.5天死亡的ATAD5 NULL小鼠不同，ATAD5无效的斑马鱼一直存活到受精后7天（DPF）。 ATAD5无效斑马鱼的致命表型在用MMS处理受精卵后孵化后就变得明显。因此，我们认为，可以使用ATAD5 NULL斑马鱼模型测试所鉴定的ATAD5变体的体内功能活性。 我们已经在体细胞癌突变中发现了一种单个序列变体，以及使用位置定向的诱变中的国际癌症基因组基因组地图集中发现的各种异位表达构建体。我们目前正在测试这些罕见的突变是否在影响ATAD5S分子功能中发现，并引起在小鼠和斑马鱼以及组织培养系统中观察到的表型。