Enhancer-mediated transcriptional dysregulation in neural crest cells and ethanol-induced teratogenesis

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Fetal Alcohol Spectrum Disorders (FASD) is among the most devastating consequences of the widespread use and abuse of alcohol. Growing evidence suggests that impairment of the differentiation, migration, and survival of neural crest cells (NCCs) is a major component of the pathogenesis of FASD. However, there is a fundamental gap in understanding how ethanol leads to the dysregulation of gene expression and subsequently impairs NCC development in embryos. Enhancers are DNA elements that govern spatial and temporal regulation of gene expression through its function as integrated transcriptional factor binding platforms. We have recently demonstrated that ethanol exposure resulted in a locus-specific reduction in enhancer activity across the genome in zebrafish embryos, as indicated by lower levels of H3K27ac mark in ethanol-exposed embryos relative to control. We termed these sites as the Ethanol-induced Variant Enhancer Loci (E-VELs). In addition, ethanol treatment reduced the activity of the selected enhancers in human NCCs (hNCCs). The objective of this proposal is to elucidate the mechanisms by which enhancers mediate ethanol-induced impairment of NCC development and teratogenesis and to establish epigenetic modulation of enhancer activity as a feasible approach for preventing FASD. The central hypothesis of this project is that ethanol epigenetically modulates the activity of the selected enhancers and subsequently disrupts the expression of their cognate genes in NCCs, leading to the impairment of the differentiation, migration, and survival of NCCs and teratogenesis and that epigenetic modulation of the activity of the selected enhancers and the expression of their cognate genes by sulforaphane (SFN) or broccoli-derived exosome-like nanoparticles (BELNs) can prevent ethanol-induced teratogenesis. To test our hypothesis, the following specific aims will be addressed: Aim 1: To identify the E-VELs that are associated with aberrant expression of their cognate genes involved in NCC and craniofacial development in hNCCs and zebrafish embryos. Aim 2: To functionally validate the role of the selected E-VELs in ethanol-induced dysregulation of gene expression, impairment of the differentiation, migration and survival of NCCs and ethanol- induced teratogenesis. Aim 3: To elucidate the mechanisms by which ethanol epigenetically modulates the activity of the selected E-VELs that modulate the expression of genes involved in the differentiation, migration, and survival of NCCs. Aim 4: To test the hypothesis that epigenetic modulation of the activity of the selected E- VELs by SFN or BELNs represents a novel therapeutic strategy for preventing ethanol-induced teratogenesis. The proposed work is innovative, because this is the first study attempting to prevent FASD through the newly recognized role of enhancers in regulating gene expression and NCC development. It also represents the first attempt to prevent FASD using edible plant-derived exosome-like nanoparticles. This study is also significant, because the results from this study are expected to provide insights into the mechanisms by which enhancers mediate ethanol-induced teratogenesis and to yield enhancer-based strategies for the prevention of FASD.