Genetic and Molecular Characterization of Dual HLH Domain Proteins in C. elegans

真核生物的发育依赖于三个精心安排的过程：分化、形态发生和生长。这些过程的协调在新受精卵的胚胎发育过程中得到了最清楚的证明，当一个细胞产生数千个分化的和形态不同的细胞类型时。在这个过程中，在整个生命周期中，发育中的生物体负责调节基因的空间和时间表达。螺旋-环-螺旋（HLH）家族转录调控因子调控了一系列广泛的发育过程。该转录因子家族的成员已在大多数真核生物系统中被发现，并且是多种关键发育过程所必需的，包括正常的胰腺功能、脑和眼的形态发生、巨噬细胞分化和骨骼肌发育。由于HLH蛋白参与了许多不同的过程，它们一直是分子遗传学家和生物化学家密切研究的焦点。已经确定了HLH蛋白的DNA结合和蛋白质二聚化活性的一般规则，并根据这些特性对HLH蛋白进行分类。在先前资助的一项试点研究中，在土壤线虫秀丽隐杆线虫中发现了一类新的HLH蛋白。鉴定出的5种蛋白各含有2个完整的HLH结构域。由于HLH蛋白已被广泛表征，因此发现两个结构域是令人惊讶的。鉴定含有一个以上HLH结构域的HLH蛋白意味着这些蛋白可能以比以前描述的更多的方式调节转录。该项目的长期目标是对含有多个HLH结构域的HLH调节因子进行体内和体外表征。为了实现这一目标，制定了以下具体目标：(1)确定五种秀丽隐杆线虫蛋白的表达模式，每种蛋白含有两个HLH结构域；(2)表征与双HLH结构域蛋白异常表达相关的表型；(3)确定两种双HLH结构域蛋白的DNA结合特性。这里描述的工作的智力价值是一个长期项目的开始，该项目侧重于HLH蛋白对真核生物发育的转录调节。一旦完成，该项目将有助于确定含有多个HLH结构域的蛋白质是否在秀丽隐杆线虫中广泛表达，以及这些蛋白质是否与具有单个HLH结构域的蛋白质表现出相同的DNA结合特性。未来的实验可以集中在这些蛋白质转录调控的体外研究上。在更广泛的范围内，这里所描述的工作将在摩根州立大学进行，这是一所HBCU，也是一所以本科生为主的城市大学。为了让更多的学生从事研究工作，本科生参与了所描述的研究的各个方面。这些学生将在当地本科生和国家研究研讨会上展示他们的研究活动。按照设计，这个项目也将允许生物系将一个活跃的研究项目整合到必修的高级本科课程中。将研究整合到本课程中，将确保摩根州立大学每位生物学专业的学生至少有一个学期的时间接触到分子遗传学的基础研究。该项目得到了分子和细胞生物科学部基因表达生物化学项目、生物科学部生物基础设施部多用户设备项目以及教育和人力资源部人力资源开发部历史黑人学院和大学本科倡议项目的支持。

Eukaryotic development depends on three carefully orchestrated processes: differentiation, morphogenesis, and growth. The coordination of these processes is most clearly demonstrated during embryological development of newly fertilized eggs, when a single cell gives rise to thousands of differentiated and morphologically distinct cell types. During this process, and throughout its life cycle, the developing organism is responsible for regulating the spatial and temporal expression of genes. The helix-loop-helix (HLH) family of transcriptional regulators regulate a wide array of developmental processes. Members of this transcription factor family have been identified in most eukaryotic systems and are required for a variety of critical developmental processes, including normal pancreatic functioning, brain and eye morphogenesis, macrophage differentiation, and skeletal muscle development. Because HLH proteins are involved in so many diverse processes, they have been the focus of intense study by molecular geneticists and biochemists. General rules for DNA binding and protein dimerization activities by HLH proteins have been determined, and the HLH proteins are classified according to these properties. During a previously funded pilot study, a new class of HLH proteins was identified in the soil nematode, Caenorhabditis elegans. The five proteins identified each contained two intact HLH domains. Because HLH proteins have been extensively characterized, finding two domains was surprising. The identification of HLH proteins containing more than one HLH domain implies that these proteins may regulate transcription in more ways than previously described. The long-term goal of this project is to perform an in vivo and in vitro characterization of HLH regulators containing multiple HLH domains. To meet this goal, the following specific aims have been formulated: (1) determine expression patterns of five C. elegans proteins, each containing two HLH domains; (2) characterize phenotypes associated with aberrant expression of dual HLH domain proteins; and (3) determine the DNA binding properties of two dual HLH domain proteins.The intellectual merit of the work described here is the beginning of a long-term project that focuses on the transcriptional regulation of eukaryotic development by HLH proteins. Once completed, this project will help determine if proteins containing more than one HLH domain are widely expressed in C. elegans, and if these proteins exhibit the same DNA binding properties as those with single HLH domains. Future experiments can then focus on in vitro studies of transcriptional regulation by these proteins. On the broader scale, the work described here will be performed at Morgan State University, an HBCU that is also an urban, predominantly undergraduate university. With the goal of exposing more students to research careers, undergraduate students are involved in all aspects of the research described. These students will present their research activities at local undergraduate and at National Research Symposia. As designed, this project will also allow the Department of Biology to integrate an active research project into a required upper-level undergraduate course. The integration of the research into this course will ensure that every Biology Major at Morgan State University will have at least one semester of exposure to basic research in molecular genetics.This project is supported by the Biochemistry of Gene Expression Program in the Division of Molecular and Cellular Biosciences and the Multi-User Equipment Program in the Division of Biological Infrastructure in the Directorate for Biological Sciences, and by the Historically Black College and University Undergraduate Initiative Program in the Division of Human Resource Development in the Education and Human Resources Directorate.