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SUMO Conjugation Contributes to Immune Deviation in Nonobese Diabetic Mice by Suppressing c-Maf Transactivation of IL-4¹

Jianmei W. Leavenworth^*, Xiaojing Ma, Yin-yuan Mo^* and Mary E. Pauza^2,*,

^* Department of Medical Microbiology, Immunology, and Cell Biology and Department of Internal Medicine, Southern Illinois University School of Medicine, Springfield, IL 62702; and Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021

It is not clear why the development of protective Th2 cells is poor in type 1 diabetes (T1D). c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore, abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. In this study we demonstrate that despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblotting demonstrates that c-Maf can be modified at lysine 33 by SUMO-1 (small ubiquitin-like modifier 1). Sumoylation is facilitated by direct interaction with the E2-conjugating enzyme Ubc9 and increases following T cell stimulation. In transfected cells, sumoylation decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation assays, and enhances c-Maf localization into promyelocytic leukemia nuclear bodies. Sumoylation of c-Maf is increased in NOD CD4 cells as compared with CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation contributes to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Juvenile Diabetes Research Foundation and the Southern Illinois University Central Research Committee (to M.E.P.).

² Address correspondence and reprint requests to Dr. Mary E. Pauza, Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, 825 North Rutledge, P. O. Box 19626, Springfield, IL 62794. E-mail address: mpauza{at}siumed.edu

³ Abbreviations used in this paper: T1D, type 1 diabetes; ChIP, chromatin immunoprecipitation; c-Maf, cellular muscular aponeurotic fibrosarcoma; DN, dominant negative; HEK, human embryonic kidney 293; IL-4p, IL-4 promoter; IP, immunoprecipitation; luc, luciferase; LZD, leucine zipper domain; MARE, Maf response element; NEM, N-ethylmaleimide; NOD, nonobese diabetic; NOR, nonobese diabetes resistant; PML, promyelocytic leukemia; PML-NB, PML nuclear body; RIPA, radioimmune precipitation assay; SNP, single nucleotide polymorphism; dbSNP, SNP database; SUMO, small ubiquitin-like modifier; TAD, transactivation domain; WB, Western blotting.

⁴ The online version of this article contains supplemental material.