Identification of Essential Factors for the Alternative Lengthening of Telomeres Pathway

端粒途径选择性延长的关键因素的鉴定

• 批准号: 9891984
• 负责人: Taylor Kristine Loe
• 金额: $ 0.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891984
• 关键词: Acute Promyelocytic Leukemia; Address; Anemia; Apoptosis; Binding Proteins; Bypass; CRISPR/Cas technology; Cell Aging; Cell Line; Cells; Chromosomes; Complex; DNA; DNA Damage; DNA Repair; Ensure; Enzymes; FANCD2 protein; Fanconi Anemia Complementation Group A Protein; Genetic; Genetic Recombination; Genome Stability; Guide RNA; Human; Investigation; Knock-out; Lead; Length; Light; Maintenance; Malignant Neoplasms; Names; Nuclear; Pathway interactions; Proteins; RNA; RNA-Directed DNA Polymerase; Role; Suggestion; Telomerase; Telomere Maintenance; Telomere Pathway; Telomere Recombination; Telomere Shortening; Topoisomerase; Topoisomerase III; alternative treatment; cancer cell; endonuclease; genome editing; genome wide screen; genome-wide; homologous recombination; novel; protein complex; telomere; therapeutic target; transcription factor PML

ABSTRACT Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural ends of linear chromosomes and ensure genome stability. Due to the end replication problem, telomeres progressively shorten with each cellular division until they become critically short, resulting in cellular senescence or apoptosis. A universal feature of human cancer cells is the ability to bypass this proliferation barrier by engaging a telomere elongation mechanism, which is most often the expression of the reverse transcriptase telomerase. However, a significant portion of cancer cells (approx 10-15%) utilize a telomerase-independent pathway named the Alternative Lengthening of Telomeres (ALT) pathway that relies on homologous recombination between telomeric sequences to extend telomeres and evade replicative arrest. Cancer cells that utilize ALT are characterized by heterogeneous telomere lengths, extrachromosomal telomeric DNA and the presence of promyelocytic leukemia (PML) bodies at telomeres. Although the ALT pathway has been the subject of intense investigation, the mechanism by which it occurs is still incompletely understood. In order to shed light on the ALT pathway, we will employ CRISPR/Cas9 to generate genetic knockouts of the known ALT-associated factors PML and SUMO2 to assess their essentiality in ALT activity. Additionally, we will use CRISPR/Cas9 to perform a genome-wide gRNA knockout screen to identify novel factors required for ALT. In this way, we will identify factors essential for ALT to expand our understanding of this telomere maintenance mechanism and reveal potential therapeutic targets for the treatment of ALT cancers.

抽象的 端粒由ttaggg重复组成，这些重复由蛋白质复合物绑定，这些复合物可保护自然 线性染色体的末端并确保基因组稳定性。由于最终复制问题， 端粒逐渐缩短每个细胞分裂，直到它们变得非常短，因此 在细胞衰老或凋亡中。人类癌细胞的普遍特征是绕过的能力 这种扩散屏障通过与端粒伸长机制接合，这通常是 逆转录酶端粒酶的表达。但是，很大一部分癌细胞 （约10-15％）利用端粒酶独立的途径，称为替代性延长 端粒（ALT）途径依赖于端粒序列之间的同源重组 扩展端粒并逃避复制逮捕。利用ALT的癌细胞的特征是 异质端粒长度，外端体端粒DNA和存在 端粒的临时细胞白血病（PML）体。虽然ALT途径一直是主题 在激烈的调查中，它发生的机制仍未完全理解。为了 在ALT途径上亮了灯，我们将采用CRISPR/CAS9产生遗传敲除 已知的ALT相关因子PML和SUMO2评估其在ALT活动中的重要性。 此外，我们将使用CRISPR/CAS9执行全基因组GRNA敲除屏幕以识别 ALT所需的新因素。这样，我们将确定ALT扩展我们的重要因素 了解这种端粒维护机制，并揭示了潜在的治疗靶点 ALT癌的处理。