Developmental regulation of lens gene expression

摘要 这项研究计划的长期目标是揭示转录和转录后 哺乳动物胚胎发育过程中组织特异性基因表达调控的协调机制 透镜的发育和分化。尽管透镜的组成很简单， 成熟细胞类型，控制透镜基因的机制和途径的全范围和光谱 表达方式有待确定。透镜发育和细胞分化的标志性特征是 透镜结构所需的a-和B-/g-晶体蛋白的时间和空间调节表达， 内稳态和透明性。本申请寻求鉴定新的顺式作用DNA调控位点 以及在体内调节哺乳动物晶状体蛋白表达的新型RNA结合蛋白（RBP） 在透镜分化期间。我们现在已经将透镜中bB 3-晶状体蛋白基因的表达与晶状体上皮细胞中bB 3-晶状体蛋白的表达联系起来。 在透镜中转录因子Pax 6与其“开放”启动子染色质结构域的体内结合 预测新的顺式作用位点和转录因子及其翻译后修饰 （PTMs），其在空间和时间上控制其表达。我们还发现了瞬变核 剪接的Crybb 3 mRNA在早期透镜纤维细胞核中的积累沿着多个空间和 可能调节bB 3-和其它晶状体蛋白mRNA转运、稳定性 翻译和衰变。这些一般机制控制其他晶状体特异性基因的表达， 它们的发现是理解转录和转录后的重要的第一步 透镜分化的调节机制。为了测试我们的预测，目标1旨在 揭示晶状体特异性顺式调节语法调节小鼠Crybb 3作为模型晶体蛋白。目的 2旨在通过鉴定新的RBP来检查透镜基因表达的转录后调节 以及它们在晶状体蛋白mRNA中的位点，特别是在它们的3 '-UTR内，决定了鉴定的 分化调节的RBP，并采用最先进的MS 2-MCP系统在体内跟踪个体 晶状体蛋白mRNAs从它们在细胞核中诞生起，贯穿它们的核输出和在巨大的细胞核中定位， 单分子分辨率下透镜纤维单元间距。这些结果将揭示这些机制连接 a-、B/g-晶状体蛋白mRNA调控，鉴定与晶状体蛋白mRNA相互作用的新颖且多样的RBP， 建立Crybb 3转录的分子机制，并为3D分布提供新的见解 在透镜细胞分化的特定阶段，单个晶状体蛋白mRNA在透镜纤维细胞中的表达。我们一起努力， 这些研究将产生对所需的分子和细胞机制的变革性见解， 哺乳动物的透镜形态发生、透明度和折射率，并提供了全面的基础 了解眼睛内外更复杂组织中的基因表达。

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.