Developmental regulation of lens gene expression

晶状体基因表达的发育调控

• 批准号: 10227314
• 负责人: Ales Cvekl
• 金额: $ 48.47万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10227314
• 关键词: 3' Untranslated Regions; 3-Dimensional; Binding; Biological; Birth; Blindness; Cataract; Cell Differentiation process; Cell Nucleus; Cells; Chromatin; Code; Complex; Coupled; Crystallins; Cytoplasm; DNA; DNA Binding; DNA Modification Process; Data; Degenerative Disorder; Development; Embryo; Event; Eye; Ferritin; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic Translation; Genomics; Goals; Growth Factor; Homeostasis; Human; Individual; Knowledge; Lens Fiber; Lens development; Life Cycle Stages; Light; Link; Messenger RNA; Modeling; Molecular; Mus; Mutate; Mutation; Nuclear; Nuclear Export; Nucleic Acid Regulatory Sequences; Outcome Study; Pathway interactions; Post-Transcriptional Regulation; Post-Translational Protein Processing; Process; Proteins; Proteomics; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulatory Element; Research; Resolution; Site; Spatial Distribution; Structure; System; Testing; Tissue-Specific Gene Expression; Tissues; Transgenic Organisms; Translations; Untranslated RNA; Untranslated Regions; Visualization; base; cell type; congenital cataract; deletion analysis; design; fiber cell; gamma-Crystallins; genome wide association study; in vivo; in vivo Model; insight; lens; lens morphogenesis; novel; programs; promoter; regenerative therapy; single molecule; solute; transcription factor; transcriptomics

ABSTRACT The long-term goal of this research program is to uncover the transcriptional and posttranscriptional mechanisms coordinating the regulation of tissue-specific gene expression during mammalian embryonic lens development and differentiation. Despite the simple composition of the lens that contains only two mature cell types, the full range and spectrum of mechanisms and pathways governing lens gene expression remain to be identified. A hallmark feature of lens development and cellular differentiation is the temporally and spatially regulated expression of a- and b-/g-crystallins required for lens structure, homeostasis and transparency. This application seeks to identify novel cis-acting DNA regulatory sites and novel RNA-binding proteins (RBPs) that regulate the expression of mammalian crystallins in vivo during lens differentiation. We have now linked the expression of the bB3-crystallin gene in the lens with in vivo binding of transcription factor Pax6 to its “open” promoter chromatin domain in lens enabling prediction of novel cis-acting sites and transcription factors and their posttranslational modifications (PTMs) that spatially and temporally control its expression. We have also discovered transient nuclear accumulation of spliced Crybb3 mRNAs in early lens fiber cell nuclei along with multiple spatially and temporally regulated RBPs that likely regulate bB3- and other crystallin mRNAs transport, stability, translation and decay. These general mechanisms control expression of other lens-specific genes making their discovery an essential first step towards understanding both transcriptional and posttranscriptional regulatory mechanisms underlying lens differentiation. To test our predictions, Aim 1 is designed to uncover the lens-specific cis-regulatory grammar regulating the mouse Crybb3 as a model crystallin. Aim 2 is designed to examine posttranscriptional regulation of lens gene expression by identifying novel RBPs and their sites in crystallin mRNAs particularly within their 3’-UTRs, determining function of identified differentiation-regulated RBPs, and employing a state-of-art MS2-MCP system to in vivo track individual crystallin mRNAs from their birth in the nucleus throughout their nuclear export and localization in the vast space of lens fiber cells at single molecule resolution. These results will reveal those mechanisms linking a-, b/g-crystallin mRNA regulation, identify novel and diverse RBPs that interact with crystallin mRNAs, establish molecular mechanisms of Crybb3 transcription, and provide new insights into the 3D-distribution of individual crystallin mRNAs in lens fiber cells at specific stages of lens cell differentiation. Together, these studies will generate transformative insights into the molecular and cellular mechanisms required for mammalian lens morphogenesis, transparency and refraction and provide a basis for comprehensive understanding of gene expression in more complex tissues in and outside of the eye.

摘要 这项研究计划的长期目标是揭示转录和转录后 哺乳动物胚胎中组织特异性基因表达调控的协调机制 晶状体的发育和分化。尽管镜片的结构很简单，只包含两个 成熟细胞类型，控制晶状体基因的全范围和光谱机制和途径 表达情况仍有待确定。晶状体发育和细胞分化的一个显著特征是 晶状体结构所需的a-和b-/g-晶体蛋白的时间和空间调节表达， 动态平衡和透明度。此应用程序旨在识别新的顺式作用DNA调控位点 和调节哺乳动物体内晶体蛋白表达的新型RNA结合蛋白(RBPs) 在晶状体分化过程中。我们现在已经将晶状体中Bb3-晶体蛋白基因的表达与IN联系起来。 激活晶状体中转录因子Pax6与其“开放”启动子染色质结构域的体内结合 新的顺式作用位点和转录因子的预测及其翻译后修饰 (PTMS)在空间和时间上控制其表达。我们还发现了瞬变核 早期晶状体纤维细胞核中剪接的CrMob3 mRNAs的积聚 时间调节的限制性商业惯例可能调节Bb3-和其他晶体蛋白mRNAs的运输，稳定性， 平移和腐烂。这些一般机制控制其他晶状体特异基因的表达 它们的发现是理解转录和转录后功能的重要第一步 晶状体分化的调控机制。为了测试我们的预测，目标一号旨在 揭示晶状体特有的顺式调控语法，将小鼠Crplob3作为模型晶体蛋白进行调节。目标 2旨在通过识别新的限制性商业惯例来检测晶状体基因表达的转录后调控。 以及它们在晶体蛋白mRNAs中的位置，特别是在它们的3‘-UTRs中，决定了所识别的功能 分化调节的限制性商业惯例，并使用最先进的MS2-MCP系统在体内追踪个体 晶体蛋白mRNAs从它们在细胞核中的诞生开始，一直到它们的核输出和在广大地区的定位 单分子分辨率的晶状体纤维细胞间距。这些结果将揭示这些机制的联系 A-，b/g-晶状体蛋白mRNA调控，鉴定与晶状体蛋白mRNAs相互作用的新的和多样化的限制性商业惯例， 建立Crplob3转录的分子机制，为三维分布提供新的见解 在晶状体细胞分化的特定阶段，晶状体纤维细胞中单个晶状体蛋白mRNAs的表达。一起， 这些研究将产生对所需的分子和细胞机制的变革性见解 哺乳动物晶状体的形态发生、透明度和屈光度全面提供了基础 了解眼内外更复杂的组织中的基因表达。