Epigenetic regulation of dioxin response elements during mammalian development

我的研究计划的长期目标是了解表观遗传学在环境暴露的生物嵌入中的作用。我感兴趣的是DNA甲基化(DNaM)如何帮助细胞记住过去的暴露并影响它们未来的反应。芳香烃受体(AhR)途径是哺乳动物细胞解毒的主要代谢途径之一。它的主要配体是二恶英，它是由燃烧过程产生的，是森林火灾烟雾、内燃机和金属或石油精炼的常见产物。在刺激下，AhR通过与二恶英反应元件(DRE)结合来激活参与解毒的基因。有一些证据表明，细胞对二恶英的反应经历了启动--即第二次接触二恶英引起的反应比第一次更大，这意味着细胞保留了第一次接触的记忆。重要的是，DREs的共识结合位点包含一个CpG二核苷酸，这是dNaM在哺乳动物基因组中的主要位置。然而，我们不知道表观遗传标记是如何调节DRE的，这种启动效应是如何在表观基因组中编码的，也不知道dNaM与其他表观遗传标记相比的具体作用。我的主要假设是，dNaM改变不是诱导DRE所必需的，而是长期启动反应所必需的。为了验证这一假设，在接下来的五年里，我建议研究DRE表观遗传调控的三个具体方面。1.建立了二恶英响应过程中dNaM、开放染色质和AhR占位的动力学。首先，我们将研究在经典的二恶英TCDD刺激过程中，dNaM、AhR占位和组蛋白修饰在DRE的时间进程。2.通过表观遗传标记确定先前接触二恶英是否影响DRE的启动。在这里，我们将开发一种暴露/再暴露范式来检测启动反应的存在，并比较早期胚胎和成人细胞中的启动，看看启动是否因发育阶段而不同。3.阐明dNaM在诱导和启动二恶英反应中的具体作用。最后，我们将比较DRE中来自第一部分和第二部分的诱导和启动数据，DRE自然包含不同数量的CPGS。我们还将在不能从DRE中去除dNaM的系统中重复启动实验，以确定当dNaM水平没有变化时，是否仍在进行诱导和/或启动。这里提出的工作将为未来关于细胞反应中细胞间异质性的工作以及对影响AhR途径诱导的因素的调查奠定基础。在这个研究项目中工作的HQP将有机会在广泛的领域获得技能和经验，为他们未来的职业生涯奠定基础。

The long-term objective of my research program is to understand the role of epigenetics in biological embedding of environmental exposures. I am interested in how DNA methylation (DNAm) helps cells remember past exposures and influences their future responses. The Aryl Hydrocarbon Receptor (AhR) pathway is one of the major metabolic routes of detoxification in the mammalian cell. Its primary ligands are dioxins, which are produced by combustion processes and are common products of forest fire smoke, combustion engines, and metal or petroleum refining. Upon stimulation, AhR activates genes involved in detoxification by binding to Dioxin Response Elements (DREs). There is some evidence that the cellular response to dioxins undergoes priming - that is, a second exposure to dioxins induces a greater response than the first, which implies that the cells retain a memory of the initial exposure. Importantly, the consensus binding site for DREs contains a CpG dinucleotide, which is the primary location of DNAm in the mammalian genome. However, we do not understand how epigenetic marks regulate DREs, how this priming effect is encoded in the epigenome, nor the specific role of DNAm compared to other epigenetic marks. My overarching hypothesis is that DNAm alterations are not required for induction of DREs, but are required for long-term priming of response. To test this hypothesis, in the next five years I propose to investigate three specific aspects of epigenetic regulation of DREs. 1. Establish the kinetics of DNAm, open chromatin, and AhR occupancy during dioxin response. First, we will investigate the timing of DNAm, AhR occupancy, and histone modifications at DREs during timecourses of stimulation with TCDD, a canonical dioxin. 2. Determine whether previous exposure to dioxins influences priming at DREs though epigenetic marks. Here, we will develop an exposure/re-exposure paradigm to detect the presence of primed responses, and compare priming in cells from early embryos as well as from adults, to see whether priming differs by developmental stage. 3. Delineate the specific role of DNAm in induction and priming of the dioxin response. Finally, we will compare induction and priming data from parts 1 and 2 in DREs which naturally contain different numbers of CpGs. We will also repeat the priming experiments in a system which is incapable of removing DNAm from DREs, to determine whether induction and/or priming are still occurring when DNAm levels do not change. The work proposed here will build a foundation for future work on cell-to-cell heterogeneity in cellular responses, as well as investigation of factors which can modify induction of the AhR pathway. HQP working in this research program will have the opportunity to gain skills and experience in a broad range of areas, setting them up for success in their future careers.