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

摘要 DNA甲基化异常发生在整个癌症基因组的所有基因组背景中，并且对DNA甲基化的研究表明， 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 通过启动子超甲基化。启动子甲基化导致转录沉默， 非启动子DNA甲基化的定义很差。这种知识的缺乏严重限制了我们的能力， 了解异常DNA甲基化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建议中，我们建议调查非启动子DNA的影响， 转录组上的甲基化，并将重点研究基因3'附近的DNA甲基化的功能 形接头.使用一对同基因癌细胞（HCT 116和DKO细胞），其特异性不同在于它们的能力， 维持DNA甲基化，我们发现基因3'端差异DNA之间存在强相关性， 甲基化以及交替切割和多聚腺苷酸化（阿帕）事件。简而言之，前mRNA经历切割， 和作为正常mRNA 3'末端形成的一部分的多腺苷酸化，以及切割的替代位点， 多聚腺苷酸化可用于产生在3'非翻译区具有不同调节序列的转录物。 区域（3'UTR）或蛋白质同种型通过编码序列内的阿帕。先前的研究已经证明 癌细胞可以劫持阿帕通路，使癌基因中短的3'UTR表达偏斜， 负调控，突出阿帕作为参与癌症起始和进展的重要过程。通过 利用癌症基因组图谱（TCGA）的数据，我们还可以观察基因3'和基因4'之间的相关性。 癌症患者样本中选定基因座的DNA甲基化和阿帕。我们假设差异DNA 基因3'端的甲基化可通过调节阿帕导致促癌表达模式。在 目的1，我们将使用计算， 生物化学、分子生物学和基因组方法。在目标2中，我们将验证我们的模型和体外数据 通过在其他癌细胞系中绘制多聚腺苷酸化位点的使用并检测疾病相关的阿帕， 使用可从实验室获得的RNA-seq和DNA甲基化数据， 国际癌症基因组联盟（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，为阿帕调节提供新的机制， 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状况。

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.