Transcription Factor Cross-Talk in Developmental Toxicity

描述(由申请人提供)：在胚胎发育期间，当暴露于低水平的化学物质会扰乱发育过程并导致疾病时，对毒物的敏感性通常会增强。化学激活的转录因子作为外部毒性刺激的环境生物传感器，调节与减轻负面影响有关的基因表达。反应很少由一个转录因子来调节；相反，它通常是两个或多个转录因子之间的协调作用。其中一个可能在胚胎对毒物暴露的反应中起重要作用的相互作用是在两个配体激活的转录因子之间--芳烃受体(AHR)和核因子红系2相关因子(NRF2)。AHR和NRF2独立地调节许多基因，这些基因参与了细胞对毒物和氧化应激的反应，以及发育、凋亡和分化。由于AHR和NRF2调节不同的基因靶点，这种串扰在发育过程中的意义可能很大；然而，它们的串扰只在成年哺乳动物和细胞培养中具有特征。斑马鱼是研究发育过程中这些相互作用的一个很好的模型，因为它们作为透明胚胎快速和外部发育，可获得广泛的基因组和分子工具，以及与人类的发育信号通路的保守，这可以促进结果的推断。这项建议是为了研究使用协作和多学科的方法来阐明AHR-NRF2串扰，特别是AHR在发育过程中调节NRF2及其下游基因转录和对毒物暴露的反应中所起的作用。目的1旨在了解AHR激动剂对NRF基因表达的影响，并确定3个斑马鱼AHR基因中哪个基因调控NRF基因的表达。这将通过使用实时定量聚合酶链式反应和吗啡反义基因敲除技术进行研究。目的2确定AHRs调节NRF2和其他NRF基因表达的机制。通过生物信息学、染色质免疫沉淀和体内瞬时启动子分析，将识别参与AHR依赖的NRF表达调控的顺式调控元件。目的3确定AHR-NRF串扰对NRF2靶基因表达和诱导性的影响。这将通过使用定量逆转录-聚合酶链式反应和微阵列分析以及通过测量活性氧物种来实现。这项研究将提供对AHR与NRF相互作用的详细和机械性的理解。它将确定这种相互作用在最敏感的生命阶段-胚胎-的重要性，并提供更好的了解组合分子信号如何保护胚胎免受毒物暴露后潜在的胚胎毒性事件的影响。) 公共卫生相关性：通过使用活体动物模型，将描述两种转录因子在胚胎对环境毒物暴露的反应中的重要性。由于胚胎受到毒物影响的风险增加，了解这些转录因子在这些影响的发病机制中的作用以及帮助保护胚胎免受毒物影响的基因表达的适应性变化是很重要的。

DESCRIPTION (provided by applicant): Sensitivity to toxicants is often enhanced during embryonic development, when exposure to low levels of chemicals can disrupt developmental process and lead to disease. Chemically-activated transcription factors act as environmental biosensors of externaltoxic stimuli and regulate gene expression involved in mitigating negative effects. Rarely is the response regulated by one transcription factor; rather, it is generally a coordinated effort between two or more transcription factors. One such interaction that may play an important role in the embryonic response to toxicant exposure is between two ligand-activated transcription factors -- the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid 2-related factor 2 (NRF2). AHR and NRF2 independently regulate many genes involved in the cellular response to toxicants and oxidative stress as well as development, apoptosis, and differentiation. Because AHR and NRF2 regulate diverse gene targets, the significance of such crosstalk during development could be large; however, their crosstalk has only been characterized in adult mammals and cell culture. Zebrafish are an excellent model to study these interactions during development because of their rapid and external development as transparent embryos, the availability of extensive genomic and molecular tools, and conservation of developmental signaling pathways with humans, which can facilitate the extrapolation of results. This proposal is for research that uses a collaborative and multidisciplinary approach to elucidate AHR-NRF2 crosstalk and, in particular, the role of AHR in regulating transcription of NRF2 and its downstream genes during development and in response to toxicant exposure. Aim 1 seeks to understand the effects of AHR agonists on expression of NRF genes, and to determine which of the three zebrafish AHR genes regulate NRF expression. This will be investigated through the use of quantitative real-time PCR and morpholino antisense knockdown techniques. Aim 2 will determine the mechanism by which AHRs regulate the expression of NRF2 and other NRF genes. Through the use of bioinformatics, chromatin immunoprecipitation, and in vivo transient promoter assays, the cis- regulatory elements involved in AHR-dependent control of NRF expression will be identified. Aim 3 will determine the effect of AHR-NRF crosstalk on the expression and inducibility of NRF2 target genes. This will be accomplished by using qRT-PCR and microarray analysis, and through the measurement of reactive oxygen species. This research will provide a detailed and mechanistic understanding of the interaction of AHRs with NRFs. It will determine the importance of this interaction during the most sensitive life stage, the embryo, and provide better understanding of how combinatorial molecular signaling can protect embryos from potentially embryotoxic events following toxicant exposure. ) PUBLIC HEALTH RELEVANCE: Through the use of an in vivo animal model, the importance of two transcription factors involved in embryonic response to environmental toxicant exposure will be delineated. Because embryos are at increased risk from effects of toxicant exposure, it is important to understand the role of these transcription factors in both the pathogenesis of these effects as well as adaptive changes in gene expression that help to protect the embryo from toxicity.