IRF-1 and IRF-2 were originally discovered as the critical regulators of the interferon system; modulating the expression of the interferon- beta (IFN-beta) gene in response to viral infection. The roles of IRF-1/2 have since multiplied to include tumor-suppression (IRF-1) and oncogenic properties (IRF-2). Deletion and/or inactivation of IRF-1 has been shown to be a critical step in the development of some human leukemias. To understand how IRF-1 and IRF-2 regulate the IFN-beta gene, we propose biochemical and structural studies aimed at understanding how they recognize their target DNA sites, manifest their repression/activation activities, and how IRF-1 interacts with adjacently bound coactivators such as NK-kappaB and HMG I(Y).
The specific aims of this proposal are: (1) To characterize the structure and function of IRF-1. We have shown that IRF-1 is accommodated differently when bound to the PRD I element versus the PRD III element of the IFN-Beta promoter. This distinction is important in vivo. We will determine the structure of IRF-1 bound to the PRD III element using molecular replacement methods. We will also examine the role of residues outside of the DNA binding domain using biophysical and in vivo transfection methods. To complement these studies, we will determine the thermodynamic parameters underlying IRF-1-DNA recognition by isothermal titration calorimetry. (2) To characterize the structure and function of IRF-2. IRF-1 and IRF-2 share similarities throughout their sequence, but manifest opposing transcriptional activities. There are also unexpected differences in the DNA binding properties of the two proteins. We will determine the structure of IRF-2 bound to the PRD I element and compare it with the IRF-1/PRD I structure. We will swap regions between the two proteins in order to interchange their transcriptional activities. The transcription levels will be measured by transfection assays using reporter plasmids. (3) To determine the structural basis of transcriptional synergy between IRF-1, NF-kappaB, and HMG I(Y). We have shown that IRF-1 and NF-kappaB inhibit each other's DNA binding, but that this inhibition can be relieved by the addition of HMG I(Y). As part of our long term goal, we will characterize these complexes through analytical ultracentrifugation studies, and attempt to cocrystallize IRF-1, NF-kappaB, and HMG I(Y) as a quaternary complex. We will delete the N-terminal arm of IRF-1 in order to test our hypothesis of steric interference between IRF-1 and NF-kappaB.
Showing the most recent 10 out of 11 publications
