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.
Insulin is expressed exclusively in pancreatic beta cells, and is therefore a good example of cell-specific gene regulation. Like most genes, regulation of the insulin gene requires cooperation between multiple transcription factors. Our goal for this project has been to investigate how these factors cooperate to ensure insulin is expressed only in beta cells. To address this question, we determined crystal structures of the three transcription factors required for insulin transcription: Pdx1, E47/NeuroD and MafA. These structures not only show how these proteins recognize DNA, they also show flexibility in DNA recognition. This is an emerging theme in the field of protein-DNA recognition that is supported by data from genome-wide DNA binding studies. The flexibility in DNA binding allows these factors to regulate many genes with different combinations of transcription factors. To follow up this idea with Pdx1, we collaborated with a lab in the Department of Physics to simulate binding to DNA. This study showed that at least 2 conformations of Pdx1 are stable on the DNA: one specific and one less specific that could function in a broader range of binding sites. Another under-appreciated property that we characterized is cases in which transcription is regulated dynamically instead of reaching equilibrium. This property is referred to as kinetic regulation. We characterized two complexes that are kinetically regulated: one is the MafA-DNA complex, and the other is a complex between the cofactor DCoH and the beta cell transcription factor HNF1. MafA binds all DNA sequences with the same apparent affinity. When it finds the correct sequence, it dissociates from the DNA much more slowly. DCoH forms an inhibitory complex that is very slow to dissociate, meaning that DCoH must interact with HNF1 fast in the cell. Overall kinetic regulation suggests that transcription complexes have to be able to form only long enough to turn on a gene. If kinetic control is widespread in regulating transcription, then we must change how we think about gene expression. Competing complexes may continuously form and dissociate on genes, with increased lifetime of the right complex. Overall these studies contribute to understanding the properties of transcription factors that underlie transcriptional regulation. To further characterize how transcription factors cooperate, we have started fusing pairs of factors together and measuring their activity in cells. These new reagents promise to distinguish the gene pathways regulated by different combinations of factors. These fusion factors will become the basis for a new NSF proposal. For the educational component of my Career proposal, I developed an intensive 3-week summer class teaching molecular biology to teachers. This idea grew out of discussions with the science education department at NCSU, and the recognition that teachers do not have enough lab experience to feel comfortable in developing new labs for their classes. The capability to manipulate genes is key to most advances in molecular biology today, and students with hands-on experience in molecular biology techniques will find many opportunities to use these skills. This class also started to build bridges between the Biochemistry Department and Science Education Department at NCSU; in general these cross-disciplinary interactions are lacking. The class provides an opportunity for masters education students, and other interested teachers to develop laboratory skills. I taught the class for 3 summers to a total of 30 teachers. During the class the teachers carry out all of the steps necessary to clone the Green Fluorescent Protein from jelly fish into bacteria, generating fluorescent bacteria. Most of the teachers who took the class reported that it helped them to feel more comfortable in the lab. Many of the teachers developed labs that they brought back to their classrooms. The syllabus and a detailed curriculum are available upon request.
