The randomization of the telomerase RNA template to define the role of telomere sequence in telomere structure, function, and cellular survival

端粒酶 RNA 模板的随机化以确定端粒序列在端粒结构、功能和细胞存活中的作用

• 批准号: 9445006
• 负责人: Margaret Pruitt
• 金额: $ 0.58万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9445006
• 关键词: randomization; telomerase; RNA; template; define

 DESCRIPTION (provided by applicant): The ability to avoid replicative senescence is a hallmark of malignant transformation. At least 85% of all cancers and 90% of breast cancers acquire this capability by activating telomerase, a reverse transcriptase that uses an internal telomerase RNA template to extend chromosome ends with telomeric DNA repeats. For this reason, telomere destabilizers and telomerase inhibitors have been a central focus of anti-tumor chemotherapy. Yet, due to a limited understanding of the basic cellular processes that cause telomere fragility and due to high background noise in current cell-based assays for telomerase inhibitor screens, there has been minimal discovery of clinically relevant telomere and telomerase targeting agents. This is a study of how variant telomerase RNA templates can be used to reveal non-WT telomere sequences that are either compatible or incompatible with cellular survival. The overall hypothesis is that randomization of the template will result in the synthesis of one class of telomeric sequences that maintain core telomere functions and thereby support cell viability and a separate class that rapidly becomes uncapped by protective proteins and elicits a potent DNA Damage Response (DDR) that confers cell death. The goal of Aim 1 is to define the mechanism by which features of the Schizosaccharomyces pombe telomerase RNA (TER1) template and complementary telomere sequence can cause cellular senescence. Serial restreaks and fluorescence microscopy of DDR and repair factors will be used to achieve this aim. The resultant information will guide therapeutic strategies to uncap telomeres in cancer cells. The purpose of Aim 2 is to identify alternative TER1 sequences that can serve as a template for the synthesis of variant telomeric repeats while retaining physiologic telomere functions. The direct primer extension assay and chromatin immunoprecipitation (ChIP) will be employed to evaluate the recruitment and overall activity of variant telomerases. The ability of the telomeric proteins, Taz1 and Pot1, to bind alternative repeats will be assessed using in vitro biochemical assays coupled with ChIP. Physiologic telomere functions including protection against 3' end resection, chromosome fusions, and DDR signaling as well as gene silencing by the telomere position effect will be characterized. These experiments provide proof-of-principle that a competitive growth screen of randomized templates can reveal functional templates other than WT. Aim 3 applies this concept to human breast cancer cell lines and uses flow cytometry and Illumina sequencing to address which variant human telomerase RNA (hTR) templates confer a growth advantage versus apoptosis. The identification of alternative templates that correspond to telomere sequences distinct from WT will facilitate the development of cell-based assays that can use the synthesis of non-WT telomeres as a clear signal for telomerase activity in a background of WT telomeres.

 描述（由申请人提供）：避免复制性衰老的能力是恶性转化的标志。至少85%的所有癌症和90%的乳腺癌通过激活端粒酶获得这种能力，端粒酶是一种逆转录酶，使用内部端粒酶RNA模板以端粒DNA重复序列延伸染色体末端。因此，端粒去稳定剂和端粒酶抑制剂已成为抗肿瘤化疗的中心焦点。然而，由于对导致端粒脆性的基本细胞过程的有限理解，以及由于目前用于端粒酶抑制剂筛选的基于细胞的测定中的高背景噪声，临床相关的端粒和端粒酶靶向剂的发现很少。这是一项关于如何使用变体端粒酶RNA模板来揭示与细胞存活相容或不相容的非WT端粒序列的研究。总体假设是模板的随机化将导致一类端粒序列的合成，所述一类端粒序列维持核心端粒功能，从而支持细胞活力，并且另一类端粒序列迅速被保护性蛋白质脱帽，并引发赋予细胞死亡的有效DNA损伤反应（DDR）。目的1是确定裂殖酵母端粒酶RNA（TER1）模板和互补端粒序列的特征可以引起细胞衰老的机制。DDR和修复因子的连续再划线和荧光显微镜将用于实现这一目标。由此产生的信息将指导治疗策略，以解开癌细胞中的端粒。目的2的目的是确定替代TER1序列，可以作为模板的变体端粒重复序列的合成，同时保留生理端粒功能。将采用直接引物延伸试验和染色质免疫沉淀（ChIP）评价变体端粒酶的募集和总体活性。端粒蛋白质Taz1和Pot1结合交替重复序列的能力将使用体外生物化学测定结合ChIP进行评估。生理端粒功能，包括对3'端切除，染色体融合，DDR信号传导以及基因沉默的端粒位置效应的保护将被表征。这些实验提供了随机化模板的竞争性生长筛选可以揭示除WT之外的功能性模板的原理证明。目的3将这一概念应用于人乳腺癌细胞系，并使用流式细胞术和Illumina测序来解决哪些变体人端粒酶RNA（hTR）模板赋予相对于凋亡的生长优势。与不同于WT的端粒序列对应的替代模板的鉴定将促进基于细胞的测定的开发，所述测定可以使用非WT端粒的合成作为WT端粒背景中端粒酶活性的明确信号。