TIN2L phosphorylation and dyskeratosis congenita pathogenic variants in telomere maintenance

项目总结/摘要 本研究的目的是了解TIN 2长链异构体磷酸化的功能及其与细胞凋亡的关系。 导致先天性角化不良（DC）的致病性变体。DC是一种遗传性骨髓衰竭综合征 由端粒维持和功能的破坏引起。患者有生命危险 骨髓增生异常综合征、免疫缺陷、各种癌症、肺纤维化和肝病。 造血细胞移植解决低血细胞计数，但需要更好地理解， 治疗其他临床特征和根本原因。TINF 2中的单等位基因致病性变体（编码TIN 2） 约占DC谱系障碍的11%。以前的研究集中在两个中较短的一个 TIN 2亚型，以及TINF 2致病性变体导致DC的机制仍不清楚。TIN2 与TRF 2相互作用，TRF 2是一种直接结合端粒DNA的shelterin组分。多种DNA加工因子 通过与TRF 2相互作用靶向端粒。这些因素参与调节的长度 端粒单链3'突出端或参与端粒同源重组。单个 同源重组和过量端粒同源需要3’端DNA 重组可导致大量端粒缩短。初步数据导致假设（1） TINL的动态磷酸化调节TIN 2L和加工因子相互作用的竞争 通过改变TIN 2L构象和（2）TIN 2L改变， 磷酸化或DC相关的R282 H破坏了这种调节，导致端粒丢失， 过度切除和同源重组。为了验证这一假设，本研究首先提出敲入 内源性TIN 2L的C末端的标签。将监测标记的TIN 2L的细胞周期特异性 TRF 2和端粒的磷酸化和募集。加工因子的募集与 将评估整个细胞周期的TRF 2和端粒。接下来，它建议敲入phosphodead， 在另一个细胞系中，敲除TIN 2L并整合可诱导的TIN 2L野生型 型或TIN 2L R282 H，最常见的TIN 2 DC变体。这些细胞系将用于评价 磷酸化和R282 H对TIN 2L和加工因子与TRF 2和端粒的关联的影响。 生物化学方法将用于评估TIN 2L与加工因子对TRF 2的竞争 结合，并确定磷酸化死亡、磷酸化模拟物或R282 H突变是否改变TIN 2L的构象。 最后，使用上述细胞系的细胞和分子生物学方法将用于确定是否 这些突变导致端粒功能障碍。总之，这些拟议的研究将提供深入了解 TIN 2L在端粒生物学中的作用，并将帮助我们实现改善端粒生物学的长期目标。 了解和治疗DC和端粒生物学疾病。

Project Summary/Abstract The goal of this project is to understand the function of phosphorylation of the long isoform of TIN2 and its relation to pathogenic variants causing dyskeratosis congenita (DC). DC is an inherited bone marrow failure syndrome caused by disruption of telomere maintenance and function. Patients are at risk of life-threatening myelodysplastic syndrome, immunodeficiency, various cancers, pulmonary fibrosis, and liver disease. Hematopoietic cell transplantation addresses low blood counts, yet a better understanding will be required to treat other clinical features and the underlying cause. Monoallelic pathogenic variants in TINF2 (encodes TIN2) account for approximately 11% of DC-spectrum disorders. Previous studies have focused on the shorter of two TIN2 isoforms, and the mechanism by which pathogenic variants in TINF2 cause DC remains unclear. TIN2 interacts with TRF2, a shelterin component that directly binds telomeric DNA. Multiple DNA processing factors are targeted to telomeres through interaction with TRF2. These factors are involved in regulating the length of the telomeric single stranded 3’ overhang or are involved in telomere homologous recombination. Single stranded 3’ overhang DNA is required for homologous recombination and excess telomere homologous recombination can result in massive telomere shortening. Preliminary data leads to the hypothesis that (1) dynamic phosphorylation of TINL regulates competition of TIN2L and processing factors for interaction with TRF2 and localization to telomeres by altering TIN2L conformation and (2) TIN2L altered phosphorylation or the DC-associated R282H disrupts this regulation, resulting in telomere loss via hyperresection and homologous recombination. To test this hypothesis, this study first proposes to knock-in a tag to the C-terminus of endogenous TIN2L. Tagged TIN2L will be monitored for cell-cycle specific phosphorylation and recruitment to TRF2 and telomeres. The correlation of processing factor recruitment to TRF2 and telomeres throughout the cell-cycle will be assessed. Next, it proposes to knock-in phosphodead and phosphomimetic mutations and, in a separate cell line, to knock-out TIN2L and integrate inducible TIN2L wild type or TIN2L R282H, the most common TIN2 DC variant. These cell lines will be used to evaluate the effect of phosphorylation and R282H on the association of TIN2L and processing factors with TRF2 and telomeres. Biochemical approaches will be used to assess the competition of TIN2L with processing factors for TRF2 binding, and to determine if phosphodead, phosphomimetic, or R282H mutations alter the conformation of TIN2L. Finally, cell and molecular biological approaches using the above cell lines will be used to determine whether these mutations lead to telomere dysfunction. Together, these proposed studies will provide insight into the role of TIN2L in telomere biology, and will help us achieve the long term goal of improving the understanding and treatment of DC and telomere biology disorders.