Regulation of the telomerase RNA component in hematopoiesis

项目总结/摘要。端粒酶对健康和长寿至关重要，但有一个基本的 缺乏对它在人体细胞中如何调节的了解。这种知识差距阻碍了发展能力 用于越来越多的涉及端粒酶功能障碍的疾病谱的疗法。长期 该项目的目标是能够操纵人类细胞中的端粒酶以获得治疗益处，其中关键是 目标是造血系统。非编码端粒酶RNA组分TERC的水平是一个重要的指标。 细胞中端粒酶功能的关键决定因素。由基因突变引起的TERC水平低， 广泛的退行性疾病，包括先天性角化不良（DC），再生障碍性贫血， MDS/白血病、实体瘤、肺纤维化和肝硬化。目前尚不清楚TERC水平如何 端粒酶在细胞中受到调节，这是开发操纵端粒酶的治疗策略的重要障碍。 本提案的总体目标是了解TERC在人类干细胞中的调节方式，包括 造血干细胞（HSC）。中心假设是TERC基因座的顺式作用元件 以及其他在转录后发挥作用的因素共同决定了稳态水平 TERC在细胞中这项工作的基本原理是， 生物发生将产生一个新的理解TERC水平是如何正常调节的，这反过来将揭示 控制端粒酶活性的新方法。中心假设将通过以下两个方面进行检验： 具体目的：（1）鉴定调节人TERC表达的转录机制，和（2） 鉴定调节人类TERC水平的转录后机制。在第一个目标下，“无疤痕” 来自DC患者的诱导多能干细胞（iPS）中的基因组编辑将用于定义 新发现的人类TERC基因座中的顺式作用元件，并揭示调节TERC基因座的反式作用因子。 TERC转录。已开发并证明在联合国系统内可行的工具和技术 申请人的手将被使用。在第二个目标下，最近发现的RNA加工因子的作用， 在具有低TERC水平的DC患者中被破坏。使用患者的iPS细胞， 由申请人创建并在该基因中携带突变，转录后加工和 将研究TERC的成熟。这种方法是创新的，因为现在的新工具和见解 可用的允许焦点从端粒酶催化组分（TERT）的调节， 这一直是深入研究的主题，到同样重要但研究较少的RNA， 端粒酶的组成部分，TERC。这项研究意义重大，因为它有望产生新的 在越来越多的造血和退行性疾病中操纵端粒酶活性的策略 包括再生障碍性贫血、MDS/白血病、肺和心血管疾病，其中端粒酶 涉及功能障碍，但对于这种疾病，即使有治愈性疗法，也很少。

PROJECT SUMMARY/ABSTRACT. Telomerase is critical for health and longevity, but there is a fundamental lack of understanding of how it is regulated in human cells. This knowledge gap impedes the ability to develop therapies for a growing spectrum of disorders in which telomerase dysfunction is implicated. The long-term goal of this project is to be able to manipulate telomerase in human cells for therapeutic benefit, with a key target being the hematopoietic system. The level of the noncoding telomerase RNA component TERC is a critical determinant of telomerase function in cells. Low TERC levels resulting from genetic mutations cause a wide spectrum of degenerative disorders, including dyskeratosis congenita (DC), aplastic anemia, MDS/leukemia, solid tumors, pulmonary fibrosis, and cirrhosis. What is not known is how TERC levels are regulated in cells, which is an important barrier to developing therapeutic strategies to manipulate telomerase. The overall objective of this proposal is to understand how TERC is regulated in human stem cells including hematopoietic stem cells (HSCs). The central hypothesis is that cis-acting elements at the TERC locus and other factors that function post-transcriptionally act in concert to determine the steady-state level of TERC in cells. The rationale for this work is that deciphering how patient mutations impact TERC biogenesis will yield a new understanding of how TERC levels are normally regulated, which will in turn reveal novel approaches to manipulate telomerase activity. The central hypothesis will be tested by pursuing two Specific Aims: (1) Identify transcriptional mechanisms regulating human TERC expression, and (2) Identify post-transcriptional mechanisms regulating human TERC levels. Under the first aim, “scarless” genome-editing in induced pluripotent stem (iPS) cells from DC patients will be used to define the function of newly identified cis-acting elements in the human TERC locus, and to reveal trans-acting factors that modulate TERC transcription. Tools and techniques that have been developed and demonstrated to be feasible in the applicants' hands will be used. Under the second aim, the role of an RNA processing factor recently found to be disrupted in DC patients who have low TERC levels will be investigated. Using patient iPS cells, which have been created by the applicant and carry mutations in this gene, the post-transcriptional processing and maturation of TERC will be investigated. The approach is innovative because the new tools and insights now available allow a critical shift in focus from the regulation of the catalytic component of telomerase (TERT), which has been the subject of intense investigation, to the equally important but less well studied RNA component of telomerase, TERC. The proposed research is significant, because it is expected to yield new strategies to manipulate telomerase activity in a growing number of hematopoietic and degenerative disorders including aplastic anemia, MDS/leukemia, pulmonary and cardiovascular disease, in which telomerase dysfunction is implicated but for which there are few if any curative therapies.