Epigenetic regulation of dioxin response elements during mammalian development

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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.