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

项目摘要/摘要 端粒酶重新激活是几乎所有人类癌症发生过程中的一个基本事件。虽然 分化的成体细胞中转录沉默，其催化成分端粒酶逆转录酶 (TERT)在80%以上的人类癌症中表达。尽管最近发现重新激活TERT 启动子突变，对导致人类端粒酶重新激活的机制知之甚少 癌症。该建议旨在确定调控TERT表达的潜在分子机制。 癌症。Barthel博士此前已经证明，TERT启动子中一个区域的甲基化与 随着转录的增加。在这里，他将从功能上解决DNA甲基化导致的 重新激活癌症发展中的端粒酶(目标1)。这一目标使用表观遗传编辑来衡量 操纵DNA甲基化对端粒酶活性的影响。此外，他还将评估分子 TERT启动子甲基化影响的机制(目标2)。他将决定是否 染色质构象和转录因子(TF)结合通过甲基化来调节。最后，他会 确定DNA甲基化、转铁蛋白结合和染色质构象的变化与 端粒酶在癌症发展中的重新激活(目标3)。总的来说，建议的研究将确立 甲基化在癌症中重新激活端粒酶中的作用及其自发性的研究 重新激活。Barthel博士的长期目标是识别端粒酶重新激活的脆弱性 癌症的发展。在这个奖项的过程中，Barthel博士将得到他的主要导师Dr。 Roel Verhaak，一位著名的计算生物学家，在研究分子方面有着非凡的记录 癌症生物学。他的合作导师郑经翰博士是创新基因工程技术的先驱。 此外，巴特尔博士还组建了一个咨询委员会，成员包括：曾傑瑞·谢伊博士， 癌症中的端粒和端粒酶；染色质三维构象专家阮一军博士； 还有苏尼特·阿加瓦尔博士，他使用诱导多能干细胞来研究端粒疾病。总而言之，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动 他的专业发展计划，以促进他过渡到一个独立的学术职位。世界经济的支柱 拟议的研究技能培训计划包括基因工程、染色质方面的高级培训 构象分析和诱导多能性。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。拟议项目的工作将主要是 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 除了大学科学环境外，成功完成这些目标所必需的。鉴于他的 详细的研究计划、优秀的咨询委员会和全面的培训计划。 巴特尔将通过这一奖项迅速过渡到独立的教职员工职位。

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.