Molecular Studies on Transactivation Potential of PAX9

描述（由申请人提供）：Pax9是Pax家族的一员，该家族由9个基因编码在模式和胚胎发生中起关键作用的核转录因子。近年来，小鼠和人类基因研究已经提供了明确的证据，证明Pax9在牙列形成过程中起着关键作用。Pax9基因的功能性缺失也会导致小鼠牙齿发育停滞。最近对人类的研究发现PAX9相关突变，常染色体显性遗传形式的牙齿发育主要影响后牙。在目前报道的11个PAX9突变中，6个发生在PAX9外显子2编码的成对结构域区域。后者在介导PAX9的dna结合活性中起重要作用。然而，除了dna结合特性外，人们对Pax9作为转录调节因子的作用知之甚少。对Pax家族其他成员的研究表明，COOH末端区域具有丰富的脯氨酸、丝氨酸和苏氨酸的特征，可能介导体内的转激活功能。我们最近在一个先天性后牙缺失的大家族成员中发现了一个新的移码突变（793InsC），位于PAX9的cooh末端结构域（外显子4）。因此，了解外显子4的作用将有助于深入了解PAX9在正常和异常牙齿发育过程中功能的分子机制。我们提出了两个特定的目的来验证一个基本假设，即COOH末端包含氨基酸264 （nt793）的区域对PAX9的转激活功能至关重要，并且该区域的突变使转激活域失活，从而损害PAX9在牙齿形态发生过程中介导的靶基因的转激活。目的1将通过鉴定在细胞共转染试验中产生最大荧光素酶报告基因活性的cooh末端的最小区域来描述Pax9的转激活功能。目的2将评估793InsC/氨基酸264突变对Pax9反激活及其蛋白质结构的影响。793InsC/氨基酸264 Pax9突变的作用将通过测定793InsC突变蛋白相对于野生型的反激活变化来研究。为了补充反活化差异的生化表征，将利用圆二色性来研究793InsC/氨基酸264 Pax9突变导致的cooh末端反活化结构域的结构改变。了解Pax9反激活靶基因的机制将有助于更好地理解Pax9突变的病因学意义，并为真核细胞的转录调控提供见解。了解Pax9的生化功能最终将为研究牙齿形态发生的遗传和分子调控提供独特的工具。

DESCRIPTION (provided by applicant): Pax9 is a member of the Pax family that consists of nine genes encoding nuclear transcription factors that play key roles in patterning and embryogenesis. In recent years, mouse and human genetic studies have provided definitive evidence of a key role for Pax9 in the patterning of dentition. Functional deletion of the Pax9 gene also results in an arrest in tooth development in mice. Recent studies on humans have associated mutations in PAX9, with autosomal dominantly inherited forms of tooth agenesis that primarily affects posterior teeth. Of the 11 PAX9 mutations reported thus far, 6 occur in the region of the paired domain that is encoded by exon 2 of PAX9. The latter is important in mediating the DNA-binding activities of PAX9. Apart from DNA-binding properties, however, relatively little is known about the role of Pax9 as a transcriptional regulator. Studies on other Pax family members point to the COOH terminal region that is characteristically rich in proline, serine and threonine as potentially mediating transactivation functions in vivo. We recently identified a novel frameshift mutation (793InsC) in the COOH-terminal domain (exon 4) of PAX9 in members of a large family with congenitally missing posterior teeth. Understanding the role of exon 4 will hence shed valuable insights into the molecular mechanisms underlying PAX9 functions during normal and abnormal tooth development. Two specific aims are proposed to test the fundamental hypothesis that the region of the COOH terminal that encompasses amino acid 264 (nt793) is critical for the transactivation function(s) of PAX9 and mutations in this region inactivate the transactivation domain and compromise PAX9-mediated transactivation of target genes during tooth morphogenesis. Aim 1 will delineate the transactivation function of Pax9 by identifying the minimal region of the COOH-terminal that yields maximal luciferase reporter-gene activity in cellular co-transfection assays. Aim 2 will evaluate the consequences of the 793InsC/amino acid 264 mutation on Pax9 transactivation and its protein structure. The role of the 793InsC/amino acid 264 Pax9 mutation will be investigated by determining alterations in transactivation of the 793InsC mutant protein relative to the wild-type. To complement the biochemical characterization of the differences in transactivation, circular dichroism will be utilized to investigate structural alteration in the COOH-terminal transactivation domain resulting from the 793InsC/amino acid 264 Pax9 mutation. Knowledge of the mechanism by which Pax9 transactivate target genes will facilitate a better understanding of the etiologic significance of mutations in PAX9 as well as provide insights into the regulation of transcription in eukaryotic cells. Understanding the biochemical function of Pax9 will ultimately provide a unique tool for the study of the genetic and molecular control of tooth morphogenesis.