The IRF family of transcription factors plays critical roles in the regulation of interferons in response to viral infection, and in the development and functioning of the immune system. The IRF family includes now over ten members, characterized by a homologous DNA binding domain (DBD) at the N-terminus. We propose structural studies to explore the broader role of IRF-3 in the regulation of interferon-beta (IFN-beta) gene expression, and the role of IRF-4 in the development and functioning of the immune system. IRF-3 is activated in virally infected cells by phosphorylation of specific residues at the C-terminus, leading to nuclear translocation and binding to a so-called PRD I-III DNA element in the IFN-beta promoter. IRF-4 binds to a number of composite DNA elements in the promoters and enhancers of B-lymphoid and myeloid genes, but exclusively in association with PU.1.
Specific aims are: 1) Determine the structure of IRF-3 DBD bound to the entire PRD I-III element by crystallographic methods. 2) Determine the structure of intact, phosphorylated IRF-3 bound to the PRD I-III element. IRF-3 will be phosphorylated in vitro prior to crystallization. 3) Structurally characterize an autoinhibitory element at the N-terminus of IRF-4 by NMR methods. 4) Determine the structure of IRF-4 in complex with phosphorylated PU.I. PU.1 will be phosphorylated in vitro by casein kinase II prior to crystallization. Together, these aims explore the specificity and cooperativity of these IRFs, and the role of phosphorylation and autoinhibitory elements.
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