Regulation of LIM Homeodomain Neuroendocrine Transcription Factor Function

哺乳动物的垂体前叶是神经内分泌功能的主要器官，因为它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同位结构域组的两个转录因子Lhx3和Lhx4参与垂体前叶激素分泌细胞的规范。这些因子发挥其特定基因调控功能的生化机制尚不清楚。Lhx3存在于不同的异构体中，具有不同的dna结合和基因调控特性。本项目采用分子和生化方法来验证Lhx3是一种多位点修饰的磷酸化蛋白的假设，并且Lhx3的磷酸化调节了其基因调控特性。Lhx3将通过质谱技术进行表达、纯化和分析，并通过位点定向突变改变修饰的同种异构体。DNA结合、基因调控特性、细胞内信号通路的参与以及这些修饰的Lhx3分子的定位将使用报告基因、组成活性信号酶和共聚焦显微镜进行测试。实验还将测试Lhx4在多大程度上与Lhx3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，本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例子。研究结果的影响将超越神经内分泌学，因为LIM蛋白是动植物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这个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.