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

Morgan State University, Baltimore MD

Investigators

Abstract

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.