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，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，对有毒物质的敏感性通常会增强。化学激活的转录因子作为外界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。这种反应很少受一个转录因子的调控;相反，它通常是两个或多个转录因子之间的协调作用。在胚胎对毒物暴露的反应中可能起重要作用的一种这样的相互作用是在两种配体激活的转录因子--芳香烃受体（AHR）和核因子红细胞2相关因子2（NRF 2）之间。AHR和NRF 2独立地调节参与细胞对毒物和氧化应激的反应以及发育、凋亡和分化的许多基因。由于AHR和NRF 2调节不同的基因靶点，因此这种串扰在发育过程中的意义可能很大;然而，它们的串扰仅在成年哺乳动物和细胞培养中得到表征。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，以及与人类的发育信号通路的保护，这可以促进结果的外推。这项建议是研究，使用协作和多学科的方法来阐明AHR-NRF 2串扰，特别是AHR在调节转录的NRF 2及其下游基因在发展过程中的作用，并在响应有毒物质暴露。目的1研究AHR激动剂对斑马鱼NRF基因表达的影响，并确定哪一个AHR基因调控NRF的表达。这将通过使用定量实时PCR和吗啉代反义敲低技术进行研究。目的二是阐明AHRs调控NRF 2及其他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NRF表达的AHR依赖性控制的顺式调节元件。目的3研究AHR-NRF串扰对NRF 2靶基因表达和诱导的影响。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。这项研究将提供一个详细的和机制的理解AHRs与NRFs的相互作用。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，并更好地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。) 公共卫生关系：通过使用体内动物模型，两个转录因子参与胚胎对环境毒物暴露的反应的重要性将被划定。由于胚胎受到毒物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适应性变化中的作用非常重要。