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，当暴露于低水平的化学物质会破坏发育过程并导致疾病时。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，并调节与减轻负面作用有关的基因表达。由一个转录因子调节的响应很少。相反，这通常是两个或多个转录因子之间的协调努力。在两个配体激活的转录因子之间，可能在胚胎对毒性暴露的胚胎反应中起重要作用的一种相互作用 - 芳基烃受体（AHR）和核因子红细胞2相关因子2（NRF2）。 AHR和NRF2独立调节了许多与细胞对毒物反应以及氧化应激以及发育，凋亡和分化的基因。由于AHR和NRF2调节了多种基因靶标，因此在发育过程中这种串扰的重要性可能很大。但是，它们的串扰仅在成年哺乳动物和细胞培养中才得以表征。斑马鱼是在开发过程中研究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对发展信号通路的保护，这可以促进结果的外推。该建议是针对使用协作和多学科方法来阐明AHR-NRF2串扰的研究，尤其是AHR在调节NRF2转录及其在开发过程中的下游基因的作用，并响应毒剂暴露。 AIM 1试图了解AHR激动剂对NRF基因表达的影响，并确定三个斑马鱼AHR基因中的哪个调节NRF表达。这将通过使用定量实时PCR和吗啡反义敲低技术来研究。 AIM 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态启动子测定，将确定与AHR依赖性控制NRF表达相关的顺式调节元素。 AIM 3将确定AHR-NRF串扰对NRF2靶基因的表达和诱导性的影响。这将通过使用QRT-PCR和微阵列分析以及通过测量活性氧来实现。这项研究将对AHR与NRF的相互作用提供详细的机械理解。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，并更好地了解组合分子信号传导如何保护胚胎免受毒性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，将描述两个转录因素对环境毒物暴露的反应的重要性。由于胚胎受到毒物暴露的影响的风险增加，因此重要的是要了解这些转录因子在这些作用的发病机理中的作用以及基因表达的自适应变化，从而有助于保护胚胎免受毒性的影响。