Molecular mechanisms of early tooth development

描述（由申请方提供）：转录因子在细胞命运决定和正常器官发育所必需的组织特异性基因表达过程中发挥重要作用。我们前期的研究表明Msx同源盒基因家族编码的转录因子在外胚层器官的发育过程中起着重要的作用。对小鼠的遗传研究表明，Msx基因家族的第一个成员Msx 1的功能丧失会影响牙齿的形成。我们报告说，（i）Msx 1突变小鼠表现出牙齿发育失败，并在发育的早期阶段（称为芽期）停滞，（ii）Msx 1控制几个下游基因的表达，这些基因在牙齿发育的芽期与Msx 1共表达。我们目前的初步研究表明，（i）Msx 1在体外和体内与几种转录因子相互作用，这些转录因子在牙齿发育的芽期与Msx 1共表达;（ii）Msx 1在体内选择性地与特定的靶基因启动子结合;（iii）Msx 1在体内通过SUMO化在翻译后水平被Sumo 1修饰，（iv）Sumo 1与Msx 1在芽期牙齿间充质中共表达，以及（v）由Sumo 1引起的Msx 1的sumoylation改变了Msx 1与牙齿特异性蛋白质-蛋白质相互作用网络及其靶基因选择相互作用的能力。这些观察结果作为我们提出的假设的基础，即Msx 1转录因子在早期牙齿发育过程中的分子功能取决于上下文依赖性，牙齿特异性转录因子网络和翻译后修饰的组合作用。为了解决这一假设，该提议具有以下研究目标：（a）在体外和体内验证Msx 1相互作用蛋白伴侣（B）确定Msx 1靶启动子如何在活细胞中受Msx 1和Msx 1相互作用蛋白复合物的组合作用调节（c）研究Msx 1的类小泛素化如何有助于发育中牙齿中基因的调节，通过分析Msx 1依赖的蛋白质-蛋白质相互作用网络和Msx 1对靶基因的选择是否受到Msx 1的类小泛素化或非类小泛素化状态的调节。对Msx 1调控蛋白功能的基本了解将为研究Msx 1在正常条件下的功能、调控早期牙齿发育的转录机制、综合征型或非综合征型缺牙的发病机制提供有价值的见解，并可能为这些疾病的治疗和牙齿再生提供有价值的信息。这项研究也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一个潜在的一般机制。 公共卫生相关性：该项目旨在了解Msx 1功能的分子机制，使用发育中的牙齿作为模型系统。为了实现这一目的，当代分子生物学技术将被用来测试所提出的假设，即Msx 1转录因子在早期牙齿发育过程中的分子功能取决于上下文依赖性，牙齿特异性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.