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

胎儿酒精谱系障碍(FASD)是广泛使用的最具破坏性的后果之一 以及酗酒。越来越多的证据表明，分化、迁移和存活的损害 神经脊细胞(NCCs)是FASD发病机制的重要组成部分。然而，有一个基本的 对乙醇如何导致基因表达失调并随后损害NCC的认识存在差距 胚胎发育。增强子是控制基因的空间和时间调节的DNA元件 通过其作为整合转录因子结合平台的功能进行表达。我们最近做了 证实了酒精暴露导致整个基因组中基因位点特异性增强子活性的降低。 在斑马鱼胚胎中，酒精暴露的胚胎中H3K27ac标记的水平低于 控制力。我们将这些位点称为乙醇诱导变异增强子基因座(E-VELS)。此外，乙醇 治疗降低了人类NCC(HNCC)中选定的增强子的活性。这项提议的目的是 目的是阐明增强剂介导乙醇诱导的NCC发育损害的机制 并建立增强基因活性的表观遗传调节作为一种可行的方法 预防FASD。该项目的中心假设是乙醇在表观遗传学上调节细胞的活性。 所选择的增强子随后扰乱它们的同源基因在NCC中的表达，导致 神经干细胞分化、迁移和存活的障碍及致畸和表观遗传学 萝卜硫素对所选增强子活性及其同源基因表达的调控 (SFN)或西兰花衍生的外切体样纳米颗粒(BELN)可以防止乙醇诱导的畸形。至 测试我们的假设，将解决以下具体目标：目标1：确定 与NCC和颅面发育相关的同源基因异常表达 HNCC和斑马鱼胚胎。目的2：从功能上验证选定的E-VEL在乙醇诱导中的作用 基因表达失调，NCC分化、迁移和存活障碍，以及乙醇- 诱发畸形。目的3：阐明乙醇表观遗传调控的机制。 所选E-VEL的活性，这些E-VEL调节与分化、迁移、 以及NCC的生存。目的4：检验表观遗传调控所选E蛋白活性的假设 SFN或BELN的VELS是预防乙醇致畸的一种新的治疗策略。 这项拟议的工作具有创新性，因为这是第一项试图通过新的 认识到增强子在调节基因表达和NCC发展中的作用。它也代表着第一个 尝试使用可食用的植物来源的外切体样纳米颗粒来预防FASD。这项研究也具有重要意义， 因为这项研究的结果有望为增强剂的作用机制提供见解 介导乙醇诱导的致畸作用并产生基于增强子的预防FASD的策略。

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.