All MHC class II genes are regulated by a set of conserved upstream elements, of which the central X-box elements (X1 and X2) have been shown to be required during all aspects of regulation. We have shown that X2BP and RFX form coorperative complexes with X-box DNA from the HLA-DRC MHC class II gene, implicating that X2BP is the class II MHC X2-box factor. This observation suggests a solution to a paradox that existed for several years, involving the failure of RFX to bind to all X boxes of the MHC class II gene family. We hypothesize that efficient binding of RFX to X-box DNA reguires cooperative binding with X2BP, thereby functionally reducing the in vitro consequences of minor sequence differences between X boxes. This hypothesis will be tested. Until recently little was known about X2BP. We showed that X2BP purifies as two polypeptides of 46 and 120 kDa. The identification of the 120 kDa subunit has yet to be elucidated. The goals of this study are to characterized fully the X2BP factor and the genes that encode it. It is anticipated that the completion of this project will provide a rigorous genetic and biochemical model of class II MHC gene expression in B cells. Such a model, including the ability to control class II expression by cAMP levels, may lead to the development of specific reagents to control the ability of cells to express MHC class II products during immune responses during infection, autoimmune disease, and organ transplantation. Experiments are designed specifically to: 1) Clone the genes encoding X2BP; 2) Analyze the structure and function of X2BP genes and proteins; 3) Characterize X2BP protein-protein interactions; 4) Determine of changes in cAMP levels that influence MHC class expression function through X2BP.
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