Growth factor independence-1 (Gfi-1), a multi-zinc finger transcription factor, is a vital protein that lies at the core of an intricate signaling network that regulates a wide variety of cellular processes, including lymphopoiesis, myeloipoiesis, nervous system development, cell proliferation and differentiation, and restriction of hematopoietic stem cell (HSC) self-renewal. This multi-functional nature, in particular the seemingly opposite effects in promoting T-cells and restricting HSC, has been attributed to its ability to recognize a variety of protein partners. Ultimately, the results of such protein-protein interactions are dependent on its concomitant interactions with nucleic acid through a zinc binding domain. This zinc domain of Gfi-1 is highly unusual both in demonstrating a potentially novel mode of nucleic acid recognition, and simultaneously interacting with multiple protein binding partners. As a multi-domain protein, Gfi-1 can be readily divided into smaller domains such that the domain functions can be dissected and investigated individually, then integrated to gain an understanding of the function of the whole protein. Detailed structural knowledge at atomic resolution of the interaction between Gfi-1 and its target DNA or its associating protein partners will provide insights into mechanisms of specific DNA recognition, as well as important protein-protein interactions by zinc fingers. The following four specific aims will be addressed: 1) Determine the structure of free Gfi-1 zinc finger 3-5 and characterize inter-finger dynamics; 2) Determine the structure of Gfizf35 bound to a 16-mer consensus DNA; 3) Characterize binding of Gfi-1 zinc fingers with different DNAs by multiple biophysical and NMR methods; 4) Map the interaction between Gfi-1 and ETO transcriptional co-repressor. Success of these studies will provide a model system for rationalizing transcription repression by a non-SNAG-containing homolog family, and provide results that will serve as a paradigm for understanding how transcriptional regulation by a repressor can afford different physiological outcomes based on different protein-protein and protein-nucleic acid interactions.
This project will focus on the concomitant protein-nucleic acid and protein-protein interactions of Gfi-1 transcription factor. Detailed structural information derived from this research will provide new insight into the mechanism of how regulation of gene transcription is mediated by a variety of protein-protein interaction involving otherwise known as canonical DNA binding motifs. By tackling well-defined hypothesis, as those laid out in this proposal, trainees will learn the process of scientific investigation and simultaneously master the use of multidisciplinary approaches for addressing structure-function relationships in macromolecules.
