Transcription Factor Cross-Talk in Developmental Toxicity

发育毒性中的转录因子交叉对话

• 批准号: 8231978
• 负责人: Larissa Williams
• 金额: $ 4.12万
• 依托单位: WOODS HOLE OCEANOGRAPHIC INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231978
• 关键词: AHR gene; ARNT gene; Adult; Agonist; Animal Model; Antioxidants; Antisense Oligonucleotides; Apoptosis; Aromatic Hydrocarbons; Aryl Hydrocarbon Receptor; Bioinformatics; Biological Assay; Biosensor; Cell Culture Techniques; Cell physiology; Cessation of life; Chemicals; Computer Simulation; Dependence; Development; Developmental Process; Disease; Dose; Elements; Embryo; Embryonic Development; Enzymes; Equilibrium; Event; Exhibits; Exposure to; Family; GSTP1 gene; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genomics; Helix-Turn-Helix Motifs; Human; In Situ Hybridization; Lead; Life; Ligands; Mammalian Cell; Mammals; Measurement; Measures; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Oxidants; Oxidative Stress; Paper; Pathogenesis; Pathway interactions; Physiology; Play; Promoter Regions; RNA; Reactive Oxygen Species; Regulation; Regulatory Element; Reporter; Research; Response Elements; Risk; Rodent; Role; Series; Signal Pathway; Signal Transduction; Staging; Stimulus; Stress; Techniques; Testing; Tetrachlorodibenzodioxin; Time; Toxic Environmental Substances; Toxic effect; Toxicant exposure; Transcriptional Regulation; Xenobiotics; Zebrafish; activating transcription factor; bZIP Domain; base; chromatin immunoprecipitation; combinatorial; in vivo; interdisciplinary approach; knock-down; loss of function; mRNA Expression; member; nuclear factor-erythroid 2; promoter; public health relevance; receptor; receptor binding; response; tool; toxicant; transcription factor

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.

描述（由申请人提供）：在胚胎发育过程中，对毒物的敏感性通常会增强，此时接触低水平的化学物质会破坏发育过程并导致疾病。化学激活的转录因子充当外部毒性刺激的环境生物传感器，并调节参与减轻负面影响的基因表达。反应很少由一种转录因子调节；相反，它通常是两个或多个转录因子之间的协调努力。一种可能在胚胎对毒物暴露的反应中发挥重要作用的相互作用是两个配体激活的转录因子——芳基碳氢化合物受体（AHR）和核因子红细胞2相关因子2（NRF2）之间的相互作用。 AHR 和 NRF2 独立调节许多参与细胞对毒物和氧化应激反应以及发育、凋亡和分化的基因。由于 AHR 和 NRF2 调节不同的基因靶标，因此发育过程中这种串扰的重要性可能很大。然而，它们的串扰仅在成年哺乳动物和细胞培养物中得到表征。斑马鱼是研究发育过程中这些相互作用的绝佳模型，因为它们作为透明胚胎的快速和外部发育、广泛的基因组和分子工具的可用性，以及与人类发育信号通路的保护，这可以促进结果的外推。该提案适用于使用协作和多学科方法来阐明 AHR-NRF2 串扰的研究，特别是 AHR 在发育过程中和响应有毒物质暴露时调节 NRF2 及其下游基因转录的作用。目标 1 旨在了解 AHR 激动剂对 NRF 基因表达的影响，并确定斑马鱼的三个 AHR 基因中哪一个调节 NRF 表达。这将通过使用定量实时 PCR 和吗啉代反义敲除技术进行研究。目标 2 将确定 AHR 调节 NRF2 和其他 NRF 基因表达的机制。通过使用生物信息学、染色质免疫沉淀和体内瞬时启动子测定，将鉴定参与 AHR 依赖性 NRF 表达控制的顺式调控元件。目标 3 将确定 AHR-NRF 串扰对 NRF2 靶基因表达和诱导性的影响。这将通过使用 qRT-PCR 和微阵列分析以及活性氧的测量来实现。这项研究将对 AHR 与 NRF 的相互作用提供详细和机制的理解。它将确定这种相互作用在最敏感的生命阶段（胚胎）中的重要性，并更好地理解组合分子信号传导如何保护胚胎免受有毒物质暴露后潜在的胚胎毒性事件的影响。 ） 公共卫生相关性：通过使用体内动物模型，将描述参与胚胎对环境毒物暴露反应的两种转录因子的重要性。由于胚胎受到有毒物质暴露影响的风险增加，因此了解这些转录因子在这些影响的发病机制以及有助于保护胚胎免受毒性影响的基因表达的适应性变化中的作用非常重要。