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

胎儿酒精谱系障碍（FASD）是广泛使用酒精的最具破坏性的后果之一。 和酗酒越来越多的证据表明，分化，迁移和存活的损害 神经嵴细胞（NCCs）的凋亡是FASD发病机制的主要组成部分。然而，有一个基本的 在理解乙醇如何导致基因表达失调并随后损害NCC方面存在差距 胚胎发育。增强子是控制基因的时空调控的DNA元件 通过其作为整合的转录因子结合平台的功能表达。我们最近 表明乙醇暴露导致基因组中增强子活性的位点特异性降低 在斑马鱼胚胎中，如暴露于乙醇的胚胎中H3 K27 ac标记的水平较低所示， 控制我们将这些位点称为乙醇诱导的变体增强子位点（E-VEL）。此外，乙醇 治疗降低了人NCC（hNCC）中所选增强子的活性。本提案的目的 阐明增强子介导乙醇诱导的NCC发育受损的机制 和致畸作用，并建立增强子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 选择的增强子，并随后破坏其同源基因在NCC中的表达，导致 NCC的分化、迁移和存活的损害以及致畸作用和表观遗传学 通过萝卜硫素调节所选增强子的活性及其同源基因的表达 (SFN)或西兰花衍生的外泌体样纳米颗粒（BELN）可以防止乙醇诱导的致畸作用。到 为了检验我们的假设，将解决以下具体目标：目标1：确定 与参与NCC和颅面发育的同源基因的异常表达相关， hNCC和斑马鱼胚胎。目的2：功能上验证所选的E-VEL在乙醇诱导的细胞凋亡中的作用。 基因表达失调，NCC的分化、迁移和存活受损，以及乙醇- 诱导致畸目的3：阐明乙醇表观遗传调节 所选的E-VEL调节参与细胞分化、迁移 和净捐助国的生存。目的4：为了检验所选E-受体活性的表观遗传调节是一个假设， 通过SFN或BELN的VEL代表了预防乙醇诱导的致畸作用的新的治疗策略。 这项工作是创新的，因为这是第一项试图通过新的方法来预防FASD的研究。 增强子在调节基因表达和NCC发展中的公认作用。它也代表了第一个 尝试使用可食用植物来源的外泌体样纳米颗粒预防FASD。这项研究也很有意义， 因为这项研究的结果有望提供对增强剂 介导乙醇诱导的致畸作用，并产生用于预防FASD的基于增强子的策略。

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.