Molecular mechanisms of early tooth development

描述（由申请人提供）：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的表达中发挥重要作用。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Msx1的功能缺失会影响牙齿的形成。我们报道了(i) Msx1突变小鼠表现出牙齿发育失败并在早期发育阶段（称为芽期）停滞；（ii） Msx1控制了几个下游基因的表达，这些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) Msx1在体外和体内与几个在牙齿发育芽期与Msx1共表达的转录因子相互作用（ii） Msx1在体内选择性地结合到特定的靶基因启动子上，（iii） Msx1在翻译后水平上被Sumo1修饰，在体内被Sumo1、（iv） Sumo1在牙芽期间质中与Msx1共表达；(v) Sumo1对Msx1的Sumo1化修饰改变了Msx1与牙齿特异性蛋白-蛋白相互作用网络及其靶基因选择的相互作用能力。这些观察结果为我们提出的假设提供了基础，即Msx1转录因子在早期牙齿发育过程中的分子功能取决于环境依赖性、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，本研究计划有以下研究目标：(a)在体外和体内验证Msx1相互作用蛋白伙伴(b)确定Msx1靶启动子如何通过Msx1和Msx1相互作用蛋白复合物(s)在活细胞中的组合作用进行调节(c)研究Msx1的summoylation如何有助于调节发育中的牙齿中的基因。通过分析Msx1的susuylated或non- susuylated状态是否调节Msx1依赖性蛋白-蛋白相互作用网络和Msx1的靶基因选择。对Msx1调节蛋白功能的基本理解将为了解Msx1在正常情况下的功能、调节早期牙齿发育的转录机制、综合征型或非综合征型牙齿畸形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。该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。

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.