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

 描述(由申请人提供)：避免复制衰老的能力是恶性转化的标志。至少85%的癌症和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，它使用内部的端粒酶RNA模板来延长染色体末端的端粒DNA重复。正因为如此，端粒不稳定剂和端粒酶抑制剂一直是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了解有限，以及当前用于端粒酶抑制物筛选的基于细胞的检测中的高背景噪声，临床上相关的端粒和端粒酶靶向制剂的发现很少。这是一项关于如何使用不同的端粒酶RNA模板来揭示与细胞生存相容或不相容的非WT端粒序列的研究。总体假设是，模板的随机化将导致合成一类端粒序列和一类单独的端粒序列，端粒序列维持核心端粒功能，从而支持细胞存活，而另一类端粒序列迅速不受保护性蛋白质的限制，并引发强大的DNA损伤反应(DDR)，从而导致细胞死亡。目标1的目的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导致细胞衰老的机制。将使用DDR和修复因子的连续重排和荧光显微镜来实现这一目标。由此产生的信息将指导治疗策略，以揭开癌细胞端粒的帽子。目的2的目的是确定可替代的TER1序列，该序列可用作合成不同端粒重复序列的模板，同时保留生理端粒功能。直接引物法和染色质免疫沉淀(CHIP)将被用来评估不同的端粒酶的招募和整体活性。端粒蛋白Taz1和POT1结合可选重复序列的能力将通过体外生化分析与芯片相结合来评估。本课程将介绍端粒的生理功能，包括对3‘端切除、染色体融合、DDR信号的保护以及端粒位置效应引起的基因沉默。这些实验提供了原理证明，随机模板的竞争性增长筛选可以揭示WT以外的功能模板。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测序来研究哪些变异的人端粒酶RNA(HTR)模板具有相对于凋亡的生长优势。识别与不同于WT的端粒序列相对应的可选模板将有助于基于细胞的分析的发展，该方法可以利用非WT端粒的合成作为WT端粒背景中端粒酶活性的明确信号。

 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.