Molecular mechanisms of early tooth development

描述(由申请人提供)：转录因子在决定细胞命运和表达正常器官发育所必需的组织特异性基因方面发挥着重要作用。我们先前的研究表明，MSX同源框基因家族编码的转录因子在外胚层器官的发育中起着重要的作用。对小鼠的遗传学研究表明，MSX基因家族的第一个成员Msx1的功能丧失会影响牙齿的形成。我们报道：(I)Msx1突变小鼠在牙齿发育的早期阶段表现出牙齿发育失败和停滞，称为芽期，(Ii)Msx1控制着几个下游基因的表达，这些下游基因在牙齿发育的芽期与Msx1共表达。我们目前的初步研究表明：(I)在牙齿发育的萌芽阶段，Msx1在体外和体内与几个与Msx1共表达的转录因子相互作用；(Ii)Msx1在体内选择性地与特定的靶基因启动子结合；(Iii)在翻译后水平上，Msx1在体内通过SUMO1被SUMO1修饰；(Iv)在芽期牙间充质中，SUMO1与Msx1共表达；(V)SUMO1通过SUMO1改变Msx1与牙齿特异的蛋白质相互作用网络及其目标基因选择的能力。这些观察结果为我们提出的假设提供了基础，即Msx1转录因子在牙齿发育早期的分子功能取决于上下文相关的牙齿特异转录因子网络和翻译后修饰的组合作用。为了解决这一假说，该建议有以下研究目标：(A)验证MSX1在体内外与蛋白质的相互作用伙伴；(B)确定在活细胞中MSX1目标启动子如何受MSX1和MSX1相互作用蛋白复合体(S)的联合作用调节；(C)通过分析MSX1依赖于MSX1的蛋白质相互作用网络和目标基因选择是否受到MSX1总和或非总和状态的调节，研究MSX1在发育中对基因的调控作用。对Msx1调控蛋白功能的基础了解将有助于深入了解Msx1在正常条件下的功能、调控牙齿早期发育的转录机制、综合征型和非综合征型缺牙的发病机制，并可能为这些疾病的治疗和牙齿再生提供有价值的信息。这项研究还将为理解细胞特异性转录因子网络如何实现靶基因选择提供潜在的一般机制。 公共卫生相关性：该项目旨在以发育中的牙齿为模型系统，了解Msx1发挥作用的分子机制。为了达到这一目的，现代分子生物学技术将被用来检验所提出的假设，即Msx1转录因子在牙齿发育早期的分子功能取决于上下文相关的牙齿特异转录因子网络和翻译后修饰的组合作用。

DESCRIPTION (provided by applicant): Transcription factors play important roles during cell fate determination and expression of tissue-specific genes that are necessary for normal organ development. Our previous studies revealed that transcription factors encoded by the Msx homeobox gene family play an important role in the development of ectodermal organs. Genetic studies in mice indicate that loss-of-function of the first member of the Msx gene family, the Msx1, affects tooth formation. We reported that (i) the Msx1 mutant mice exhibit a failure of tooth development and arrest at an early stage of development known as the bud stage and (ii) that Msx1 controls the expression of several downstream genes that are co-expressed with Msx1 during the bud stage of tooth development. Our current preliminary studies indicate that (i) Msx1 interacts in vitro and in vivo with several transcription factors that are co-expressed with Msx1 during bud stage of tooth development (ii) that Msx1 selectively binds in vivo to a specific target gene promoter, (iii) that Msx1 is modified at the posttranslational level by sumoylation in vivo, by Sumo1, (iv) that Sumo1 is co-expressed with Msx1 in the bud stage dental mesenchyme and that (v) Msx1 sumoylation by Sumo1 modifies the ability of Msx1 to interact with the tooth-specific, protein-protein interaction network and its target gene selection. These observations serve as the basis for our proposed hypothesis, that the molecular function of Msx1 transcription factor during early tooth development depends on the combinatorial action of a context dependent, tooth-specific transcription factor network and posttranslational modifications. To address this hypothesis, this proposal has the following research objectives (a) To validate the Msx1-interacting protein partners in vitro and in vivo (b) To determine how the Msx1 target promoter is regulated by the combinatorial action of Msx1 and Msx1-interacting protein complex(s) in living cells (c) To investigate how sumoylation of Msx1 contributes to regulation of genes in the developing tooth, by analyzing whether the Msx1- dependent protein-protein interaction network and target gene selection by Msx1 is modulated by the sumoylated or non-sumoylated state of Msx1. The fundamental understanding of how Msx1 regulatory protein functions will provide valuable insight on the function of Msx1 under normal conditions, on the transcriptional mechanism regulating early tooth development, on the pathogenetic mechanisms of syndromic or non-syndromic types of anodontia, and may provide valuable information towards treatment of these diseases and tooth regeneration. This study will also provide a potential general mechanism for understanding how cell-specific network of transcription factors achieve target gene selection. PUBLIC HEALTH RELEVANCE: This project aims at understanding the molecular mechanism by which Msx1 functions, using the developing tooth, as a model system. To accomplish this purpose, techniques of contemporary molecular biology will be employed to test the proposed hypothesis that the molecular function of Msx1 transcription factor during early tooth development depends on the combinatorial action of a context dependent, tooth-specific transcription factor network and posttranslational modifications.