BIOCHEMICAL ANALYSIS OF PITX2 AND TOOTH DEVELOPMENT

患有Rieger综合征的患者表现为与新型Pitx 2同源框转录因子基因突变相关的牙齿发育不全。Pitx 2为研究牙齿形成的分子控制提供了独特的工具，因为它在牙齿发育的最早阶段选择性表达。然而，调控Pitx 2表达和功能的机制尚不清楚。本申请的重点是了解Pitx 2在牙齿形态发生中的分子/生化机制。基于初步数据，Pitx 2是bicoid样同源框基因家族的成员，具有转录反式激活活性。我们建议确定Pitx 2的功能域和测试我们的假设，即C-末端39个氨基酸作为一个DNA结合抑制区调节蛋白质-蛋白质相互作用。我们已经表明，Pitx 2的转录活性是由垂体特异性POU同源域转录因子，Pit-1。在牙齿形态发生中，很可能需要额外的因子来完成Pitx 2的反式激活活性，类似于发育中的垂体中的Pit-1。本申请的一个主要目标是回答这个问题。什么是调节Pitx 2功能的因素？我们建议首先建立Pitx 2功能域的工作模型。在这种情况下，我们将问如何C-末端区域抑制DNA结合和反式激活，以及如何自然发生的Rieger突变影响Pitx 2功能？在第一组实验中，我们将询问Pitx 2活性是否由与口腔上皮转录因子的物理相互作用控制，正如我们使用Pit-1蛋白的初步研究所建议的那样。我们将测试与牙齿上皮中与Pitx 2共表达的已知候选物的相互作用，例如Msx 2和Dlx 2同源结构域蛋白。这种方法也将使我们能够识别新的相互作用蛋白质。然后，我们将测试这些蛋白质相互作用对Pitx 2功能很重要的假设。我们将使用最小的bicoid增强子/TK启动子和明显的靶基因（含有多个bicoid样元件的Dlx 2启动子）来测试Pitx 2活性. Dlx 2在Pits 2之后的发育中的牙齿原基中表达，并且似乎是Pitx 2调节的极好候选者。在第二组实验中，我们将确定Rieger综合征突变和磷酸化对Pitx 2活性的影响。这些研究将为更好地理解早期牙齿发育的分子控制提供基础。

Patients with Rieger syndrome, present with dental hypoplasia associated with mutations in the novel Pitx2 homeobox transcription factor gene. Pitx2 provides a unique tool for studying the molecular control of tooth formation since it is selective expressed at the earliest stage of tooth development. However, the mechanisms that regulate Pitx2 expression and function are not known. The focus of this application is to understand the molecular/biochemical mechanisms of Pitx2 in tooth morphogenesis. Based on preliminary data, Pitx2 is a member of the bicoid-like homeobox gene family and has transcriptional transactivation activity. We propose to identify Pitx2 functional domains and test our hypothesis that the C-terminal 39 amino acids act as a DNA binding inhibitory region modulated by protein-protein interactions. We have shown that Pitx2 transcriptional activity is modulated by the pituitary specific POU homeodomain transcription factor, Pit-1. In tooth morphogenesis it is likely that additional factors are clearly needed for full Pitx2 transactivation activity, similar to Pit-1 in the developing pituitary. A major objective of this application will be to answer the question., what are the factors that regulate Pitx2 function? We propose to first establish a working model of Pitx2 functional domains. In this context we will ask how does the C-terminal region inhibit DNA binding and transactivation, and how do the naturally occurring Rieger mutations affect Pitx2 function? In the first set of experiments, we will ask if Pitx2 activity is controlled by physical interactions with oral epithelial transcription factors, as suggested by our preliminary studies using the Pit-1 protein. We will test interactions with known candidates that are co-expressed with Pitx2 in the dental epithelia, such as the Msx2 and Dlx2 homeodomain proteins. This approach will also enable us to identify novel interacting proteins. We will then test the hypothesis that these protein interactions are important for Pitx2 function. We will test Pitx2 activity using a minimal bicoid enhancer/TK promoter and with an apparent target gene, the Dlx2 promoter which contains multiple bicoid- like elements. Dlx2 is expressed in the developing tooth primordia after Pits2 and appears to be an excellent candidate for regulation by Pitx2. In the second set of experiments we will determine the effect of the Rieger syndrom mutations and phosphorylation on Pitx2 activity. These studies will provide the foundation for a better understanding of the molecular control of early tooth development.