Molecular Studies on Transactivation Potential of PAX9

PAX9 反式激活潜力的分子研究

• 批准号: 7281045
• 负责人: HITESH KAPADIA
• 金额: $ 2.25万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7281045
• 关键词: animal genetic material tag; beta galactosidase; bioassay; cell line; circular dichroism; dental development; dentition; developmental genetics; epithelium; fibroblasts; frameshift mutation; luciferin monooxygenase; mesenchyme; polymerase chain reaction; proline; protein structure function; reporter genes; serine; site directed mutagenesis; threonine; transcription factor; transfection

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.

描述(申请人提供)：Pax9是Pax家族的成员之一，该家族由9个基因组成，编码在模式形成和胚胎发育中发挥关键作用的核转录因子。近年来，小鼠和人类的遗传学研究已经提供了确凿的证据，证明Pax9在牙列模式中起着关键作用。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了PAX9的相关突变，常染色体显性遗传形式的牙齿发育不全主要影响后牙。到目前为止报道的11个PAX9突变中，有6个发生在PAX9外显子2编码的配对结构域区域。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。对其他Pax家族成员的研究表明，COOH末端区域富含脯氨酸、丝氨酸和苏氨酸，可能在体内介导反式激活功能。我们最近在一个先天缺失后牙的大家族中发现了PAX9基因COOH末端结构域(外显子4)的一个新的移码突变(793InsC)。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的分子机制。提出了两个特定的目标来检验这一基本假设，即COOH端的第264位氨基酸(Nt793)区域对于PAX9的反式激活功能(S)至关重要，该区域的突变使反式激活结构域失活，并破坏了PAX9在牙齿形态发生过程中介导的靶基因的反式激活。目的1通过鉴定细胞共转染实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的反式激活功能。目的2将评估793 InsC/氨基酸264突变对Pax9反式激活及其蛋白结构的影响。793 InsC/氨基酸264 Pax9突变的作用将通过确定793InsC突变蛋白相对于野生型反式激活的变化来研究。为了补充反式激活差异的生化特征，将利用圆二色谱来研究793InsC/氨基酸264 Pax9突变导致的COOH末端反式激活结构域的结构变化。了解Pax9反式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，并为真核细胞中转录调控提供见解。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控制提供独特的工具。