Global profiling of O-GlcNAcylated c-Rel-dependent gene expression

Abstract

Abstract NF-κB c-Rel is a critical regulator of transcription in T cells. We previously showed that c-Rel is O-GlcNAcylated at a single residue, serine 350, and this posttranslational modification has a dual role in positive and negative regulation of c-Rel-dependent transcription. Here we performed a targeted RNA expression study to examine how c-Rel O-GlcNAcylation influences the transcriptional landscape of known NF-κB-dependent genes in T cells. We found groups of genes that were upregulated, downregulated or unregulated upon increased O-GlcNAcylation, and several of these genes were dependent on c-Rel serine 350 as analyzed by mutational studies. We also developed a novel site-specific antibody targeting serine 350 O-GlcNAcylated c-Rel. Our functional studies show that this novel antibody is capable of detecting hyperglycemia-induced c-Rel O-GlcNAcylation in cells isolated from diabetes patients and identify O-GlcNAcylation-dependent DNA binding of c-Rel by chromatin immunoprecipitation. Knowledge gained from the gene expression study and the use of S350 site-specific anti-O-GlcNAcylated c-Rel antibody have the potential to reveal the role of c-Rel O-GlcNAcylation in regulating T cell function in autoimmune diabetes.