Understanding the full spectrum of epigenetic vulnerability in cancer through the delineation of DNA methylation function in gene 3' end

通过描绘基因 3 端 DNA 甲基化功能，全面了解癌症的表观遗传脆弱性

• 批准号: 10334486
• 负责人: Angela H Ting
• 金额: $ 38.93万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334486
• 关键词: 3' Untranslated Regions; Affect; Binding; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; CCCTC-binding factor; Cancer Biology; Cancer Patient; Cancer cell line; Candidate Disease Gene; Cell Line; Cells; Chemicals; Code; Collection; Colon; Coupled; DNA; DNA Methylation; DNA Modification Process; DNA Sequence; DNA-Binding Proteins; DU145; Data; Defect; Disease; Distal; Dynein ATPase; Epigenetic Process; Event; Exhibits; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genomic approach; Genomics; Goals; HCT116 Cells; Human; Hypermethylation; In Vitro; International; Knowledge; LNCaP; Light; Location; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methylation; MicroRNAs; Modeling; Modification; Molecular; Molecular Biology; Mutate; Nuclear; Oncogenes; Pathologic; Pattern; Physiological; Polyadenylation; Polyadenylation Pathway; Prevalence; Prevention strategy; Process; Prostate; Protein Isoforms; Proteins; Proteome; RNA Polymerase II; RNA Processing; Regulation; Reporter; Reporting; SW480; Sampling; Seminal; Shapes; Site; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Transcript; Tumor Suppressor Genes; base; cancer cell; cancer genome; cancer initiation; cancer therapy; cancer type; chromatin immunoprecipitation; epigenetic regulation; genome-wide; improved; in vivo; mRNA Precursor; methylation pattern; novel; predictive modeling; prevent; promoter; side effect; stable cell line; therapy design; transcription factor; transcription termination; transcriptome; transcriptome sequencing; treatment strategy; tumor progression; tumorigenesis

Abstract DNA methylation abnormalities occur in all genomic contexts throughout the cancer genome, and studies of such aberrant epigenetic patterns have led to seminal discoveries regarding tumor suppressor gene silencing by promoter hypermethylation. While promoter hypermethylation causes transcriptional silencing, the functions of non-promoter DNA methylation are poorly defined. Such lack of knowledge severely limits our ability to contextualize the effects of abnormal DNA methylation on cancer biology and to realize the full potential of epigenetic-based cancer therapy. In this proposal, we propose to investigate the impact of non-promoter DNA methylation on the transcriptome and will focus on studying the functions of DNA methylation near gene 3' ends. Using a pair of isogenic cancer cells (HCT116 and DKO cells) that differ specifically in their ability to maintain DNA methylation, we discovered a robust association between gene 3' end differential DNA methylation and alternative cleavage and polyadenylation (APA) events. Briefly, pre-mRNAs undergo cleavage and polyadenylation as part of normal mRNA 3' end formation, and alternative sites of cleavage and polyadenylation can be utilized to produce transcripts with varying regulatory sequences in the 3' untranslated regions (3' UTRs) or protein isoforms via APA within coding sequences. Previous studies have demonstrated that cancer cells can hijack the APA pathway to skew expression of short 3' UTRs in oncogenes to evade negative regulation, highlighting APA as an important process involved in cancer initiation and progression. By leveraging the Cancer Genome Atlas (TCGA) data, we could also observe the correlation between gene 3' DNA methylation and APA at select loci in cancer patient samples. We hypothesize that differential DNA methylation in gene 3' ends can result in cancer-promoting expression patterns through regulation of APA. In Aim 1, we will define the mechanism of DNA methylation-regulated APA using a combination of computational, biochemical, molecular biology, and genomic approaches. In Aim 2, we will validate our model and in vitro data by mapping polyadenylation site usage in additional cancer cell lines and testing for disease-relevant APA events across different cancer types using publically available RNA-seq and DNA methylation data from the International Cancer Genome Consortium (ICGC). The results of our study will improve overall functional understanding of non-promoter DNA methylation, provide a novel mechanism for APA regulation, and ultimately accelerate discovery of novel targets for cancer management. We also envision that our findings here can have broad impact on our knowledge of how epigenetic regulation shapes the transcriptome in cancer as well as in other human healthy and pathological conditions.

摘要 DNA甲基化异常发生在整个癌症基因组的所有基因组环境中，并且研究 这种异常的表观遗传模式导致了关于肿瘤抑制基因沉默的开创性发现 通过启动子的超甲基化。虽然启动子超甲基化导致转录沉默，但其功能 非启动子DNA甲基化的定义不明确。这种知识的缺乏严重限制了我们 了解DNA甲基化异常对癌症生物学的影响，并充分发挥 基于表观遗传学的癌症治疗。在这个提案中，我们建议研究非启动子dna的影响。 转录组的甲基化，将重点研究基因3‘附近DNA甲基化的功能。 结束了。使用一对相同基因的癌细胞(HCT116和DKO细胞)，它们的能力特别不同 维持DNA甲基化，我们发现基因3‘端差异DNA之间有很强的相关性 甲基化和交替切割和多腺苷(APA)事件。简而言之，前信使核糖核酸经历切割 和作为正常的mRNA3‘末端形成的一部分的聚腺苷酸化，以及裂解和 多聚腺苷可以被用来产生在3‘未翻译的区域具有不同调控序列的转录本 编码序列中通过APA的区域(3‘UTRs)或蛋白质异构体。之前的研究已经证明 癌细胞可以劫持APA途径，扭曲癌基因中短3‘UTRs的表达，从而逃避 负调控，强调APA是参与癌症发生和发展的一个重要过程。通过 利用癌症基因组图谱(TCGA)数据，我们还可以观察到基因3‘之间的相关性。 癌症患者样本中特定基因座DNA甲基化和APA的研究我们假设差异DNA 基因3‘端的甲基化可以通过调节APA而导致促癌的表达模式。在……里面 目标1，我们将使用计算和分析相结合的方法来定义DNA甲基化调节APA的机制， 生物化学、分子生物学和基因组学方法。在目标2中，我们将验证我们的模型和体外数据 通过绘制更多癌细胞系中多聚腺苷酸化位点的使用图和检测与疾病相关的APA 使用可公开获得的RNA-seq和DNA甲基化数据在不同癌症类型中的事件 国际癌症基因组联合会(ICGC)。我们的研究结果将改善整体功能 了解非启动子DNA甲基化，为APA调控提供了一种新的机制，并 最终加速发现癌症管理的新靶点。我们还设想我们的发现 这可能会对我们关于表观遗传调控如何塑造转录组的知识产生广泛的影响 癌症以及其他人类健康和病理条件下的癌症。