Regulation of LIM Homeodomain Neuroendocrine Transcription Factor Function

哺乳动物垂体前叶是一个重要的神经内分泌器官，分泌多种重要的激素。 在发育过程中，被称为转录因子的蛋白质调节编码这些激素的基因的激活。 LIM同源结构域组的两个转录因子Lhx3和Lhx4参与垂体前叶中的垂体分泌细胞的特化。 关于这些因子发挥其特定基因调控功能的生化机制知之甚少。 Lhx3以不同的亚型存在，具有不同的DNA结合和基因调控特性。 该项目使用分子和生物化学方法来测试Lhx3是一种在多个位置被修饰的磷蛋白，并且Lhx3的磷酸化调节其基因调控特性的假设。 Lhx3将通过质谱技术表达、纯化和分析，并且修饰的同种型将通过定点突变而改变。 DNA结合，基因调控特性，参与细胞内信号传导途径，和本地化这些修改Lhx3分子将使用报告基因，组成型活性信号传导酶和共聚焦显微镜进行测试。 实验还将测试Lhx4与Lhx3在多大程度上具有相似但不同的转录特性。 基因组学的一个重要问题是了解有限数量的基因如何介导复杂生物体的发育，这项研究展示了如何从单个基因产生多种蛋白质以及它们的功能如何依赖于转录后调控的一个例子。 结果将产生超出神经内分泌学的影响，因为LIM蛋白是动物和植物中许多其他系统的发育和功能所必需的。对本科生和研究生的培训和指导也是该项目的一个组成部分。

The mammalian anterior pituitary gland is a major organ for neuroendocrine function because it secretes multiple, important hormones. During development, proteins known as transcription factors regulate activation of the genes that encode these hormones. Two transcription factors of the LIM homeodomain group, Lhx3 and Lhx4, are involved in the specification of the hormone-secreting cells in the anterior pituitary. Very little is known about the biochemical mechanisms by which these factors exert their specific gene regulatory functions. Lhx3 exists in different isoforms, which have distinct DNA-binding and gene regulatory properties. This project uses molecular and biochemical approaches to test the hypothesis that Lhx3 is a phosphoprotein that is modified at multiple locations, and that phosphorylation of Lhx3 modulates its gene regulatory properties. Lhx3 will be expressed, purified and analyzed by mass spectrometry techniques, and modified isoforms will be changed by site-directed mutatgenesis. The DNA binding, gene regulatory properties, involvement of intracellular signaling pathways, and localization of these modified Lhx3 molecules will be tested using reporter genes, constitutively active signaling enzymes and confocal microscopy. Experiments also will test to what extent Lhx4 has similar, but distinct, transcriptional properties to Lhx3. An important issue from genomics is to understand how a limited number of genes can mediate the development of complex organisms, and this study shows an example of how multiple proteins can be generated from individual genes, and how their functions depend on post-transcriptional regulation. Results will have an impact beyond neuroendocrinology, because LIM proteins are required for development and function of many other systems in both animals and plants. Training and mentoring of undergraduate and graduate students also are an integral part of this project.