CAREER: Structural Studies of Basic-helix-loop-helix and Homeodomain Interactions that Elicit Cell-specific Gene Expression

Cell-specific gene expression is fundamental to the functions of differentiated cells in multi-cellular organisms. What restricts gene expression to specific cell types is not well understood. Transcription factors regulate gene transcription by binding DNA sequences near a gene, called the promoter sequence. Generally combinations of factors are required to express a gene. Often these factors are members of large transcription factor families with limited DNA binding selectivity in themselves. This project investigates the role of protein-protein interactions between transcription factors in regulating promoter recognition and gene activation. The project focuses on interactions between two large transcription factor families, basic-helix-loop-helix (bHLH) and homeodomain (HD) factors. Different bHLH and HD factors interact in a number of different cell types. As a model system, the regulation of the proopiomelanocortin (POMC) promoter will be studied. This promoter regulates cell-specific expression in pituitary corticotrophs. Corticotrophs are one of six endocrine cells of the pituitary, each secreting a unique hormone. A region of the POMC promoter, called a 'minienhancer', binds the bHLH heterodimer, E47/NeuroD1, and the pituitary HD factor Pitx1. The bHLH and HD domains interact in the absence of DNA. In addition, the POMC promoter binds the corticotroph-restricted T-box domain protein T-pit. This research will test the hypothesis that complementarity between transcription factor interactions and the minienhancer sequence contributes to cell-specific activation of POMC. Aim 1 is to determine whether the same protein-protein interactions between factors in solution are maintained when bound to the minienhancers. Aim 2 and 3 will develop structural models of the protein and DNA interactions of the POMC minienhancer using a combination of high-resolution crystal structures and small angle X-ray and neutron scattering. Aim 4 will test the validity of the models and the contribution of the protein-protein interactions to DNA binding and transcriptional activation by generating mutations in protein interfaces.

The current study will contribute to our understanding of transcription factor interactions to the 'promoter code' that controls cell-specific gene expression. bHLH and HD factors play key roles in differentiation and development, yet distinguishing features of individual family members remain elusive, even within the same cell. This research will serve as the basis for future studies addressing the relationship between promoter architecture and stabilization of the transcription factor complexes by further interactions. The combination of protein crystallography and small angle scattering (in collaboration with Oak Ridge National Labs) promises to facilitate structural studies of larger multi-protein complexes.

The interdisciplinary approach of this project provides an excellent context to involve students in authentic research, and promote inquiry-based teaching approaches. A new class entitled Biology Research as Inquiry, co-listed between the Departments of Biochemistry and Mathematics, Science & Technology Education, will engage education masters students in research. The class will use the research project to motivate teaching scientific content with the goal of modeling inquiry-based teaching approaches. Class participants will develop laboratory skills in molecular biology and apply those skills to generate mutations to test the validity of the minienhancer model (Aim 4 of the research plan). At the same time they will be challenged to understand and consider the research hypothesis. A follow-up class will support teachers in applying the research experience to their high school classrooms. Equipment will be made available for teachers to borrow. The project will provide a context for involving biochemistry undergraduates in research. A Department of Education research assistant will help implement the educational goals of the project and act as a liaison for teachers and schools. Minority students in the education and biochemistry programs will be encouraged to participate.