The Role of MTF-1 as a Mediator of Ocular Toxicity

 描述（由申请人提供）：眼部病变，如白内障和黄斑变性，是世界上视力受损和失明的主要原因。虽然这些病理形成为衰老的自然过程，但有几个环境和生活方式因素会增加这些疾病的风险，例如吸烟，暴露于光和紫外线辐射，氧化应激，类固醇治疗和应激诱导的DNA损伤。我们建议研究金属应答转录因子1（MTF-1）在参与眼发育的一系列基因的转录调控中的作用，这些基因也可能导致环境应激相关的眼病理。该提案的主要目标是确定MTF-1在正常眼睛发育中起什么作用，以及眼睛中细胞应激对MTF-1信号传导的不适当激活是否会导致促进眼部病变的基因的错误表达。斑马鱼胚胎和成体将被用作我们的模式生物，并且将在人原代透镜上皮细胞系中进行比较实验。通过原位杂交，实时PCR和通过RNA-seq的全局转录组学分析进行的靶向基因表达评估将用于确定MTF-1激活或抑制对眼睛特异性基因调控的影响。我们选择有毒金属镉作为我们的模型环境风险因素，因为它是MTF-1信号传导的最知名的激活剂之一，并且因为它被认为是吸烟者白内障和黄斑变性风险增加的直接致病因素。与微阵列表达谱互补的ChIP-Seq分析将用于确定MTF- 1的DNA结合，以确认在调节负责眼睛发育和应激相关病理的基因中的直接作用。通过瞬时转染试验和染色质免疫沉淀的额外启动子表征将用于最终确认MTF-1在斑马鱼和人类中对眼睛特异性基因的调节。从这些实验中获得的结果将产生公共卫生的意义，通过提供新的洞察力的作用机制，环境压力有助于眼部病变。除了发现涉及眼睛发育的潜在新调控途径以及对白内障发生和黄斑变性的新见解外，拟议研究的成功完成将进一步扩大斑马鱼目前作为眼科疾病相关研究的模式生物的用途。

 DESCRIPTION (provided by applicant): Eye pathologies, such as cataracts and macular degeneration, are the leading causes of impaired vision and blindness in the world. Although these pathologies form as a natural process of aging, there are several environmental and lifestyle factors that increase the risk of these diseases, such as cigarette smoking, exposure to light and ultraviolet radiation, oxidative stress, steroid treatment and stress-induced DNA damage. We propose to investigate the role of the metal-responsive transcription factor 1 (MTF-1) in the transcriptional regulation of a suite of genes involved in eye development that may also contribute to environmental stress- related eye pathologies. The main objectives of this proposal are to determine what role MTF-1 plays in normal eye development and whether or not inappropriate activation of MTF-1 signaling by cellular stress in the eye can lead to misexpression of genes that promote eye pathologies. Zebrafish embryos and adults will be used as our model organism and comparative experiments will be performed in a human primary lens epithelial cell line. Targeted gene expression assessments by in situ hybridization, real-time PCR and global transcriptomic profiling via RNA-seq will be used to determine the effects of MTF-1 activation or inhibition on the regulation of eye-specific genes. We have chosen the toxic metal cadmium as our model environmental risk factor because it is one of the best known activators of MTF-1 signaling and because it is believed to be a direct causative factor in the increased risk of cigarette smokers to cataractogenesis and macular degeneration. ChIP-Seq analysis complementary to the microarray expression profiling will be used to determine DNA binding by MTF- 1 as confirmation of a direct role in the regulation of genes responsible for eye development and stress-related pathologies. Additional promoter characterization via transient transfection assays and chromatin immunoprecipitation will be used for final confirmation of MTF-1 regulation of the eye-specific genes in zebrafish and humans. The results obtained from these experiments will yield public health significance by providing novel insight into mechanisms of action by which environmental stressors contribute to eye pathologies. In addition to the discovery of a potential new regulatory pathway involved in eye development with new insight into cataractogenesis and macular degeneration, successful completion of the proposed research will further expand the current use of zebrafish as a model organism for eye disease-related research.