Epigenetic alterations underlying tumorigenic potential of Glioblastoma Cells

描述(申请人提供)：多形性胶质母细胞瘤(GBM)几乎普遍是致命的，从最初诊断时起中位生存期为12-18个月，需要新的治疗方法。表观遗传学和遗传学在癌症发生的所有阶段都相互合作，包括在脑瘤中。表观遗传机制围绕着核小体，核小体是由组蛋白核心和包裹在组蛋白核心周围的DNA组成的结构。多种表观遗传机制共同作用，产生促进或抑制基因激活的染色质状态。这些机制包括DNA甲基化(在CpG位点)、组蛋白变体和修饰以及核小体定位。染色质状态的全基因组研究主要集中在表观遗传调控的个别方面，因此，共同决定转录状态的表观遗传调控机制的完整图谱尚未完成。使用我一直在开发的一种新技术(gnome-seq)，我将在正常人类神经前体细胞和GBM细胞的同一DNA链中生成DNA甲基化和核小体定位的全基因组地图。这将识别异常沉默的基因，这些基因需要DNA去甲基化和染色质重塑才能重新激活。这些数据将与H_2A变异体H_2A.Z的定位图谱相结合。除了存在于重要的基因组调节区，如增强子和绝缘子，H_2A_Z还定位于活性和稳定的基因启动子。结合DNA甲基化、核小体占有率和H2A.Z定位数据，将识别哪些基因组座位是活跃的、稳定的和沉默的，从而提供详细描述GBM中存在的异常染色质结构的关键信息，这对表观遗传治疗具有重要意义。有了这些信息，在独立的阶段，我将用DNA去甲基化试剂5-aza-cdr处理GBM细胞，并测量去甲基化恢复正常基因表达模式和分解异常染色质结构的能力。除了测量DNA甲基化、核小体占有率和H2A.Z定位外，我还将通过测量神经球的形成来评估肿瘤形成的潜力。在该奖项的指导阶段，我将生成DNA甲基化、核小体占有率和H2A.Z定位的全基因组图谱，这些图谱将描述正常人类神经前体细胞和GBM细胞的表观遗传学图景。我还将学习分析全基因组表观基因组数据所必需的新的生物信息学技术，并参与有助于在学术研究机构获得并成功担任独立职位的职业发展机会。相关性：基底膜肿瘤很难治疗，对这些细胞的表观遗传学图景知之甚少。了解这些细胞的表观遗传学变化以及它们对DNA去甲基化治疗的反应将对未来的治疗具有重要意义。在这里，我将研究几个水平的表观遗传调控，以及它们在脑瘤中是如何改变的。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is almost universally fatal with median survival of 12-18 months from the time of initial diagnosis and new therapeutic approaches are needed. Epigenetics and genetics co-operate at all stages of carcinogenesis, including in brain tumors. Epigenetic mechanisms revolve around the nucleosome, a structure comprised of a core of histone proteins and DNA, which is wrapped around the histone core. A variety of epigenetic mechanisms work together to generate chromatin states that facilitate or inhibit gene activation. These mechanisms include DNA methylation (at CpG sites), histone variants and modifications and nucleosome positioning. Genome wide studies of chromatin states have focused on individual aspects of epigenetic regulation and as a result an integrated map of epigenetic regulatory mechanisms, which together determine transcriptional state, has not been completed. Using a novel technique that I have been developing (GNOMe-seq), I will generate genome wide maps of DNA methylation and nucleosome positioning within the same DNA strand of normal human neural progenitor cells and GBM cells. This will identify aberrantly silenced genes, which require DNA demethylation in addition to chromatin remodeling for reactivation. This data will be combined with localization profiles of the H2A variant, H2A.Z. In addition to being present at important genomic regulatory regions like enhancers and insulators, H2A.Z is localized to active and poised gene promoters. Combining DNA methylation, nucleosome occupancy and H2A.Z localization data will identify which genomic loci are active, poised and silenced, thereby providing critical information detailing aberrant chromatin structures present in GBM which has important implications for epigenetic therapy. Armed with this information, in the independent phase, I will treat GBM cells with the DNA demethylating agent 5-Aza-CdR and measure the ability of demethylation to restore normal gene expression patterns and resolve aberrant chromatin structures. In addition to measuring DNA methylation, nucleosome occupancy and H2A.Z localization, I will assess tumorigenic potential by measuring neurosphere formation. During the mentored phase of the award, I will generate the genome-wide maps of DNA methylation, nucleosome occupancy and H2A.Z localization, which will characterize the epigenetic landscape of normal human neural progenitors and GBM cells. I will also learn new bioinformatics techniques necessary for analyzing genome wide epigenomic data and participate in career development opportunities that will help in obtaining and succeeding as an independent position at an academic research institution. RELEVANCE: GBM tumors are difficult to treat, and little is known about the epigenetic landscape of these cells. Understanding the epigenetic alterations in these cells and how they respond to DNA demethylation treatment will have important implications for future therapies. Here I will investigate several levels of epigenetic regulation and how they are altered in brain tumors.