DNA demethylation and the reprogramming of transcriptional dynamics

DNA 去甲基化和转录动力学重编程

基本信息

批准号：
8875463
负责人：
Joshua Steven Bell
金额：
$ 4.31万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8875463
关键词：
Aberrant DNA Methylation Accounting Acute Affect Animal Model Antineoplastic Agents Azacitidine Breast Cancer Cell Categories Cause of Death Cell Culture Techniques Clinical Complement CpG Islands CpG dinucleotide Cytidine Cytosine DNA DNA Methylation DNA Modification Methylases DNA Modification Process DNA Polymerase II DNA Sequence DNA Sequence Alteration Data Set Deoxycytidine Drug Targeting Drug usage Dysmyelopoietic Syndromes Epigenetic Process Event Gene Activation Gene Expression Gene Expression Profile Gene Silencing Genes Genetic Transcription Genome Genomics Goals Human Hypermethylation In Vitro Island Kinetics Lead Length Licensing Link MCF7 cell Malignant Neoplasms Messenger RNA Methylation Modeling Monitor Mutation Nucleosomes Pathway interactions Pharmaceutical Preparations Polymerase Polymerase Gene Precipitation Process RNA RNA Polymerase II Refractory Research Resolution Role Second Primary Cancers Shapes Signal Transduction Site Solid Neoplasm Staging Testing Transcript Transcriptional Activation Transcriptional Regulation Tumor Suppressor Genes United States Withdrawal Work analog base bisulfite sequencing cancer cell carcinogenesis clinical efficacy demethylation design drug development drug withdrawal genome-wide genome-wide analysis global run on sequencing histone modification improved insight leukemia novel prevent promoter public health relevance research study standard of care therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Our ability to treat cancer, the second leading cause of death in the USA, remains woefully inadequate and, while novel therapies must be designed, optimization of existing therapies must be a complimentary priority. One underutilized class of chemotherapeutics is demethylating agents, currently the standard of care for several leukemias. An array of in vitro and animal model experiments have shown promise for these drugs in the treatment of solid tumors, however a limited understanding of their clinical efficacy has limited their use. Demethylating agents inhibit DNA methyltransferases, which methylate cytosines in CpG dinucleotides. CpG sites are concentrated in regions known as CpG islands, which are associated with the promoters of most human genes, are typically unmethylated, and serve as sites for transcriptional initiation. When CpG islands are methylated, the recruitment of RNA Polymerase II (Pol II) is inhibited, and the associated gene silenced. Cancer cells generally have aberrant DNA methylation profiles, including genomic hypomethylation and local hypermethylation of tumor-suppressor gene promoters. However, relief of promoter hypermethylation accounts for few transcriptional changes upon treatment with demethylating agents, and the largest portion of methylation changes in cancer occur not within CpG islands, but in flanking regions known as CpG island 'shores'. The role of DNA methylation in shores is not well established, but evidence suggests that shore methylation may regulate the release of Pol II from a paused to actively elongating state. After initiation, Pol II tracks 40-60bp before pausing, awaiting recruitment of various factors to advance to productive mRNA elongation. Studies have demonstrated a correlation between shore demethylation and enhanced transition of paused Pol II to active elongation, and in preliminary studies we have shown that Pol II pauses at shore/island transitions where methylation starkly increases. Pol II also tracks and pauses in the upstream antisense direction in a similarly regulated process referred to as divergent transcription that we hypothesize serves to prevent the spread of methylation into CpG islands. This proposal seeks to test the hypothesis that drug- induced demethylation of CpG island shores leads to the escape of RNA Polymerase II from a paused state, resulting in gene activation, and an increase in divergent transcription, both of which act to inhibit remethylation following drug withdrawal. We will test this model by treating MCF7 breast cancer cells with demethylating agents and monitoring methylation and transcriptional changes following treatment, both genome-wide, and at specific loci. We will then correlate these changes within various categories of genomic features to determine their relationship, e.g. if downstream shore hypomethylation corresponds to increased gene body transcription. These experiments will define how demethylation therapy changes transcription to inform design of novel chemotherapeutics and expand use of demethylating agents to solid tumors.

描述（由申请人提供）：我们治疗癌症（美国第二大死亡原因）的能力仍然严重不足，虽然必须设计新的疗法，但必须优先考虑优化现有疗法。一类未充分利用的化疗药物是去甲基化药物，目前是多种白血病的标准治疗方法。一系列体外和动物模型实验显示了这些药物在治疗实体瘤方面的前景，但对其临床疗效的了解有限限制了它们的使用。去甲基化剂抑制 DNA 甲基转移酶，该酶使 CpG 二核苷酸中的胞嘧啶甲基化。 CpG 位点集中在称为 CpG 岛的区域，这些区域与大多数人类基因的启动子相关，通常是非甲基化的，并作为转录起始位点。当 CpG 岛甲基化时，RNA 聚合酶 II (Pol II) 的募集会受到抑制，相关基因也会沉默。癌细胞通常具有异常的 DNA 甲基化谱，包括基因组低甲基化和肿瘤抑制基因启动子的局部高甲基化。然而，在用去甲基化剂治疗后，启动子高甲基化的缓解很少导致转录变化，并且癌症中甲基化变化的最大部分不是发生在 CpG 岛内，而是发生在称为 CpG 岛“海岸”的侧翼区域。 DNA 甲基化在 shore 中的作用尚未明确，但有证据表明 shore 甲基化可能调节 Pol II 从暂停状态到主动延伸状态的释放。启动后，Pol II 会追踪 40-60bp，然后暂停，等待各种因子的募集以推进有效的 mRNA 延伸。研究表明，海岸去甲基化与暂停的 Pol II 向主动伸长的增强转变之间存在相关性，并且在初步研究中，我们表明 Pol II 在甲基化明显增加的海岸/岛屿转变处暂停。 Pol II 还在上游反义方向上跟踪并暂停，这是一个类似的调节过程，称为发散转录，我们假设该过程有助于防止甲基化扩散到 CpG 岛中。该提案旨在检验以下假设：药物诱导的 CpG 岛岸去甲基化会导致 RNA 聚合酶 II 从暂停状态逃脱，从而导致基因激活和发散转录增加，这两者都会抑制药物后的再甲基化撤回。我们将通过用去甲基化剂处理 MCF7 乳腺癌细胞并监测治疗后全基因组和特定位点的甲基化和转录变化来测试该模型。然后，我们将在不同类别的基因组特征中关联这些变化，以确定它们的关系，例如如果下游岸的低甲基化对应于基因体转录的增加。这些实验将定义去甲基化疗法如何改变转录，从而为新型化疗药物的设计提供信息，并将去甲基化药物的用途扩大到实体瘤。