Genetic and Epigenetic Strategies for the Acquisition of Telomere Maintenance in Human Cancer Cells

项目总结/摘要 端粒酶再活化是几乎所有人类癌症发生中的基本事件。虽然 在分化的成体细胞中转录沉默，其催化组分端粒酶逆转录酶 端粒酶逆转录酶（TERT）在超过80%的人类癌症中表达。尽管最近发现重新激活端粒酶逆转录酶 启动子突变，导致端粒酶在人类中重新激活的机制知之甚少。 癌该建议旨在确定调节肿瘤细胞中TERT表达的潜在分子机制。 癌Barthel博士先前已经表明，在TERT启动子区域的甲基化与 增加转录。在这里，他将从功能上解决DNA甲基化是负责 在癌症发展中重新激活端粒酶（Aim 1）。这个目标使用表观遗传编辑来测量 操纵DNA甲基化对端粒酶活性影响。此外，他将评估分子 这是由甲基化在TERT启动子（目的2）的影响机制。他将决定是否 染色质构象和转录因子（TF）结合通过甲基化调节。最后，他会 确定DNA甲基化、TF结合和染色质构象的变化与 端粒酶再活化在癌症发展中的作用（目的3）。总的来说，拟议的研究将建立 甲基化在癌症中重新激活端粒酶中的作用，并提供对其自发性 重新激活Barthel博士的长期目标是确定端粒酶再激活的脆弱性， 癌症发展在这个奖项的过程中，巴特尔博士将得到他的主要导师，博士。 Roel Verhaak是一位著名的计算生物学家，他在研究分子生物学方面有着非凡的记录。 癌症生物学他的共同导师Albert Cheng博士开创了创新的基因工程技术。 此外，Barthel博士还组建了一个咨询委员会，其中包括： 癌症中的端粒和端粒酶;阮益军博士，三维染色质构象专家; 以及Suneet Agarwal博士，他使用诱导多能干细胞研究端粒疾病。在一起，这 多学科团队将使Barthel博士能够成功地执行拟议的实验，并推进 他的专业发展计划，以促进他过渡到一个独立的学术地位。支柱 拟议的研究技能培训计划包括遗传工程、染色质 构象分析和诱导的多能性。专业发展将包括以下关键要素 指导、赠款撰写和实验室管理。拟议项目的工作主要是 在杰克逊基因组医学实验室进行，该实验室提供所有最先进的设施 除了学院的科学环境外，成功完成目标还需要一个良好的环境。鉴于他 详细的研究计划，优秀的咨询委员会和全面的培训计划，预计博士。 巴特尔将通过这个奖项迅速过渡到一个独立的教师职位。

PROJECT SUMMARY/ABSTRACT Telomerase reactivation is a fundamental event in the genesis of nearly every human cancer. Although transcriptionally silent in differentiated adult cells, its catalytic component telomerase reverse transcriptase (TERT) is expressed in over 80% of human cancers. Despite the recent discovery of reactivating TERT promoter mutations, very little is known about mechanisms leading to the reactivation of telomerase in human cancer. This proposal aims to determine the underlying molecular mechanisms regulating TERT expression in cancer. Dr. Barthel has previously shown that methylation of a region in the TERT promoter was associated with increased transcription. Here, he will functionally resolve that DNA methylation is responsible for reactivating telomerase in cancer development (Aim 1). This Aim uses epigenetic editing to measure the impact of manipulating DNA methylation on telomerase activity. Furthermore, he will assess molecular mechanisms that are affected by methylation at the TERT promoter (Aim 2). He will determine whether chromatin conformation and transcription factor (TF) binding are regulated via methylation. Finally, he will identify what changes in DNA methylation, TF binding and chromatin conformation are associated with telomerase reactivation in cancer development (Aim 3). Collectively, the proposed studies will establish the role of methylation in reactivating telomerase in cancer and provide insights into the nature of its spontaneous reactivation. The long-term goal of Dr. Barthel is to identify vulnerabilities of telomerase reactivation during cancer development. Over the course of this award Dr. Barthel will be supported by his primary mentor, Dr. Roel Verhaak, a renowned computational biologist with a remarkable track record studying the molecular biology of cancer. His co-mentor, Dr. Albert Cheng has pioneered innovative genetic engineering technologies. In addition, Dr. Barthel has assembled an advisory committee that includes: Dr. Jerry Shay, a leader on telomeres and telomerase in cancer; Dr. Yijun Ruan, an expert on three-dimensional chromatin conformation; and Dr. Suneet Agarwal, who uses induced pluripotent stem cells to study telomere disease. Together, this multidisciplinary team will enable Dr. Barthel to successfully execute the proposed experiments and advance his professional development plan to facilitate his transition to an independent academic position. Pillars of the proposed research skills training program include advanced training in genetic engineering, chromatin conformation analysis and induced pluripotency. Professional development will feature key elements including mentorship, grant writing and laboratory management. Work towards the proposed project will primarily be conducted at The Jackson Laboratory for Genomic Medicine, which offers all the state-of-the art facilities required for the successful completion of the Aims in addition to a collegial scientific environment. Given his detailed research plan, excellent advisory committee and comprehensive training plan it is expected that Dr. Barthel will quickly transition to an independent faculty position through this award.