The AF10/DOT1 Complex, O-GlcNAc, and Transcription: From Nematodes to Mammals

Type 2 Diabetes is associated with obesity and excessive nutrient consumption and is prevalent in developed countries, including the United States with over 15 million individuals affected. The hallmark of Type 2 Diabetes is a defect in the glucose‐sensing mechanism inducing insulin resistance. Excess flux through the hexosamine biosynthesis pathway, which ultimately results in increased levels of O-linked GlcNAcylation of proteins, is recognized as one of the major contributors to insulin resistance. This modification is catalyzed by O-GlcNAc transferase (OGT) and removed by the glycosidase O-GlcNAcase (OGA). Importantly, mutations in the OGA gene have been associated with type 2 diabetes in humans. Our work in C. elegans revealed that the chromatin-binding protein ZFP-1 (AF10 in mammals) slows down transcription of highly expressed genes relevant to metabolism, such as the key insulin signaling component 3-phosphoinositide-dependent kinase 1 (pdk-1). Recently, a prevalent occurrence of O-GlcNAc modification on chromatin at the promoters of active genes was reported and implicated in transcriptional activation. In C. elegans, ZFP-1 co-localizes with O-GlcNAc genome-wide and inhibits it. The hypothesis presented here is that O-GlcNAc modification at active promoters serves to stimulate glucose-responsive transcription and that ZFP-1 and its major interacting partner DOT-1.1 act to provide a negative feedback and inhibit O-GlcNAc and transcription. To test this hypothesis, C. elegans and 3T3-L1 murine adipocytes will be used in the proposed research. In the first specific aim, the level of O-GlcNAc modification on chromatin in zfp-1 mutant will be evaluated genome-wide by ChIP-seq and the effect of O-GlcNAc on gene expression will be determined by RNA-seq analyses of zfp-1; ogt-1 and zfp-1; oga-1 double mutant worms. In the second specific aim, the chromatin localization of AF10 and O-GlcNAc in 3T3-L1 murine adipocytes will be identified by ChIP-seq. Once their overlapping target genes will be identified, expression changes of these genes upon siRNA-mediated downregulation of AF10 and modulation of O-GlcNAc levels will be analyzed by RT-qPCR.