Identifying epigenetic targets of c-myc oncogene

项目摘要：这项提案的首要目标是揭示一个以前不具特色的 C-Myc(以下简称Myc)的分子功能，是一种经常在 乳腺癌和许多其他类型的人类癌症。最近，我们发现Myc的过度表达会导致 谷氨酸-氨连接酶(GLUL)的表达升高，有趣的是，这种Myc诱导的GLUL不是 通过Myc的直接反式激活，而不是涉及GLUL基因的启动子去甲基化。我们 进一步发现，去甲基化依赖于胸腺嘧啶DNA糖基酶(TDG)的表达增加， 它是Myc基因的直接转录靶点。这些结果表明，Myc在促进 谷氨酰胺的合成，有趣的是，暗示了Myc在激活过程中的一种未知的分子功能 通过调节DNA甲基化来表达基因。这促使我们形成一个假设，即Myc可以调节 通过DNA甲基化调节基因表达。我们提出了两个具体目标来研究这一点 假设。在目标1中，我们计划通过以下方式鉴定Myc诱导的DNA甲基化和基因表达谱 全基因组亚硫酸氢盐测序(WGBS)和RNA-SEQ分析 Myc的诱导性表达或敲除。我们将首先准备不同处理的细胞培养和 提取基因组DNA和细胞总RNA用于下一代测序(NGS)。在NGS序列之后 获得读数后，我们将进行生物信息学分析，以(1)进行差异识别和注释 WGBS中的甲基化区域(DMR)，重点放在基因启动子上；(2)寻找差异表达基因 (Deg)在RNA-seq中，使用相同的DMR样本；以及(3)使用整合的 DMR和DEG的生物信息学分析，并确定优先影响的潜在生物通路 MYC通过表观遗传调控。我们期望在不同的乳房中发现Myc的特定“表观遗传靶点” 癌细胞系。在目标2中，我们计划使用传统分子来验证已鉴定的甲基化图谱 技术，并检查它们的生物学相关性。我们将首先使用定量聚合酶链式反应和焦点硫酸氢盐 对特定基因启动子进行测序，以验证在Aim 1中发现的Myc的“表观遗传靶标”。 我们还将检测TDG和Myc表观遗传靶点在不同的乳腺癌细胞系中的表达。 此外，我们将研究TDG的表达模式和通过IHC验证的表观遗传Myc靶标 使用未鉴定的乳腺癌临床组织样本，并将其与组织病理学相关联 特点和临床结果。如果成功，这个项目将发现DNA去甲基化是一种新的 Myc调控基因表达和肿瘤发生的机制。从长远来看，从 这项研究将有助于对癌症病因的理解，并为小说的发展提供启示 治疗学。

PROJECT SUMMARY: The overarching goal of this proposal is to uncover a previously uncharacterized molecular function of c-Myc (hereafter referred to as Myc), a proto-oncoprotein that is frequently amplified in breast cancer and many other types of human cancer. Recently, we found that Myc overexpression leads to elevated expression of glutamate-ammonia ligase (GLUL) and interestingly, this Myc-induced GLUL is not through the direct transactivation by Myc, rather it involves promoter demethylation of the GLUL gene. We further found that the demethylation is dependent on increased expression of thymine DNA glycosylase (TDG), which is a direct Myc transcriptional target. These results suggest an unexpected role of Myc in promoting glutamine synthesis, and intriguingly, suggest a previously unidentified molecular function of Myc in activating gene expression by regulating DNA methylation. This prompts us to form the hypothesis that Myc can regulate gene expression via the modulation of DNA methylation. We propose two Specific Aims to study this hypothesis. In Aim 1, we plan to identify Myc-induced DNA methylation and gene expression profiles by whole genome bisulfite sequencing (WGBS) and RNA-Seq using various breast cancer cell lines with stable or inducible expression or knock-down of Myc. We will first prepare cell cultures with different treatments and extract genomic DNA and total cellular RNA for the next-generation sequencing (NGS). After NGS sequence reads are obtained, we will perform the bioinformatics analysis to (1) identify and annotate differentially methylated regions (DMR) in WGBS, with focus on gene promoters; (2) identify differentially expressed genes (DEG) in RNA-seq, using the same samples for DMR; and (3) rank Myc “epigenetic targets” using integrated bioinformatics analysis of DMR and DEG, and identify potential biological pathways preferentially affected by Myc through epigenetic regulation. We expect to discover specific “epigenetic targets” of Myc in various breast cancer cell lines. In Aim 2, we plan to validate the identified methylation profiles using traditional molecular techniques and examine their biological relevance. We will first use the quantitative PCR and focal bisulfate sequencing on specific gene promoters to validate the “epigenetic targets” targets of Myc to be found in Aim 1. We will also examine the expression of TDG and the Myc epigenetic targets in various breast cancer cell lines. Furthermore, we will examine the expression patterns of TDG and validated epigenetic Myc targets by IHC using de-identified breast cancer clinical tissue samples, and correlate them with histopathological characteristics and clinical outcomes. If successful, this project will uncover DNA demethylation as a novel mechanism for Myc regulated gene expression and oncogenesis. In the long run, the knowledge gained from this study will help with the understanding of cancer etiology and shed light on the development of novel therapeutics.