The role of Lmp4 in the regulation of the cardiac transcription factor Tbx5

描述（由申请人提供）：先天性心脏病是影响人类的最普遍和最严重的疾病之一。因此，对受影响的个人、家庭和社会的有害影响强调需要更好地了解器官和细胞水平上潜在的分子事件。 T-box 基因功能改变尤其会影响心血管系统。脊椎动物基因组包含至少 18 个不同 T-box (Tbx) 基因的家族。其中，Tbx5在多种心脏谱系和结构中具有功能，人类TBX5的突变会导致霍尔特-奥拉姆综合征（HOS），一种以先天性心脏和肢体缺陷为特征的疾病。这种心脏/四肢疾病的心脏表现范围从心房和心室间隔缺损到复杂的畸形，例如法洛四联症。 HOS 表型可由 Tbx5 量减少或增加导致，表明 Tbx 蛋白水平对于正常功能至关重要。尽管它们在胚胎发生和疾病中很重要，但编码的转录因子发挥其功能的机制尚不清楚。该项目的长期目标是阐明 Tbx5 转录因子在心脏发生过程中如何在分子调控网络内相互作用，以及这些过程有时会失败的方式和原因。我们已经鉴定出一种新的 Tbx5 结合蛋白 Lmp4。我们建议测试 Lmp4 通过控制其亚细胞定位来调节 Tbx5 转录因子活性的假设。本研究的重点是研究 Tbx5/Lmp4 蛋白复合物形成对 Tbx5 核定位、Tbx5 依赖性心脏基因表达、心脏模式和形态发生的变化的作用。在细胞水平上，我们将确定 Tbx5 转录因子及其结合伴侣的定位，并询问蛋白质水平和亚细胞定位的操作是否会干扰下游基因表达。在生物体水平上，我们将在斑马鱼和鸡胚胎模型中使用互补策略。斑马鱼功能获得和丧失的实验将测试 Lmp4 在早期鱼胚胎发生中的作用，特别是心脏形成。鸡心脏中野生型和突变型结合因子的强制错误表达将研究其对心脏特异性基因表达和模式形成的作用。最终，这些研究将为 Tbx 功能提供更广泛的视角，这对于更好地了解与 Tbx5 基因突变相关的先天性心血管畸形是必要的，并将深入了解以前未知的心脏发生中转录因子调节机制。

DESCRIPTION (provided by applicant): Congenital heart defects are among the most prevalent and serious diseases affecting humans. Thus, the detrimental impact on the affected individuals, families, and society emphasizes the need for a better understanding of the underlying molecular events at the organ and cell levels. The cardiovascular system is in particular affected by altered T-box gene function. The vertebrate genome contains a family of at least 18 different T-box (Tbx) genes. Among those, Tbx5 has functions in a variety of cardiac lineages and structures, and mutations in human TBX5 result in Holt-Oram syndrome (HOS), a disease characterized by congenital heart and limb defects. The cardiac manifestations in this heart/limb disease range from atrial and ventricular septal defects to complex malformations such as tetralogy of Fallot. The HOS phenotypes can result form either reduced or increased amounts of Tbx5, indicating that the levels of Tbx proteins are critical for normal functioning. In spite of their importance in embryogenesis and disease, the mechanisms by which the encoded transcription factors exert their functions are not yet understood. The long-term objective of this project is to elucidate how the Tbx5 transcription factor interacts within a molecular regulatory network during cardiogenesis, and how and why these processes sometimes fail. We have identified a novel Tbx5 binding protein, Lmp4. We propose testing the hypothesis that Lmp4 regulates Tbx5 transcription factor activity by controlling its subcellular localization. The focus of this study is to investigate the role of Tbx5/Lmp4 protein complex formation on Tbx5 nuclear localization, changes in Tbx5-dependent cardiac gene expression, heart patterning, and morphogenesis. At the cellular level we will determine the localization of Tbx5 transcription factors and their binding partner and ask whether manipulation of protein levels and subcellular localization interferes with downstream gene expression. At the organism level we will use complementary strategies in zebrafish and chicken embryo models. Gain- and loss-of-function experiments in zebrafish will test the role of Lmp4 in early fish embryogenesis with a special emphasis on heart formation. Forced misexpression of wild type and mutated forms of the binding factor in chicken hearts will investigate its role on cardiac-specific gene expression and pattern formation. Ultimately, these studies will provide a broader perspective of Tbx function, necessary for a better understanding of congenital cardiovascular malformations associated with Tbx5 gene mutations, and will provide insight into a previously unknown mechanism for transcription factor regulation in cardiogenesis.