Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc)

Abstract

O-linked N-acetylglucosamine transferase (OGT) reversibly modifies serine and threonine residues of many intracellular proteins with a single beta-O-linked N-acetylglucosamine residue (O-GlcNAc), and has been implicated in insulin signaling, neurodegenerative disease, cellular stress response, and other important processes in mammals. OGT also glycosylates RNA polymerase II and various transcription factors, which suggests that it might be directly involved in transcriptional regulation. We report here that the Drosophila OGT is encoded by the Polycomb group (PcG) gene, super sex combs (sxc). Furthermore, major sites of O-GlcNAc modification on polytene chromosomes correspond to PcG protein binding sites. Our results thus suggest a direct role for O-linked glycosylation by OGT in PcG-mediated epigenetic gene silencing, which is important in developmental regulation, stem cell maintenance, genomic imprinting, and cancer. In addition, we observe rescue of sxc lethality by a human Ogt cDNA transgene; thus Drosophila may provide an ideal model to study important functional roles of OGT in mammals.

Fig. 1.

Schematic map of the Drosophila Ogt gene and sxc mutations. The solid boxes represent exons with black (protein coding) and white (5′ and 3′ noncoding) regions. The angled lines below indicate the introns and their sizes. The nature and position of sxc mutations is indicated above the lines; X indicates a stop codon.

Fig. 2.

Measurements of OGT protein levels and enzyme activity. (A) Western blot analysis of homogenates of larvae containing 2 functional copies of the sxc gene (wild type, lane1), 1 copy (sxc²⁶³⁷/Balancer (Bal), lane 2), or no copies (sxc²⁶³⁷/sxc²⁶³⁷ homozygous, lane 3). (B) OGT enzymatic activity in extracts from wild-type, sxc²⁶³⁷/Bal, and sxc²⁶³⁷/sxc²⁶³⁷ larvae were assayed using recombinant p62 as the acceptor substrate. Activity is reported as a percentage of activity relative to that found for wild-type fly larvae extracts. (C) Western blot analysis of homogenates of wild-type flies (lane 1) versus those carrying a tubulin-GAL4 driven Ogt RNAi transgene (lane 2). (D) Western blot analysis of adult wild-type (lane 1) and sxc³/sxc²⁶³⁷ mutant flies rescued by a tubulin-GAL4 driven human Ogt transgene (lane 2). For Western blots, all extracts were probed for OGT (Upper) using rabbit anti-OGT Ab (H-300) and also for β-tubulin using a mouse anti-β-tublin mAb (E7) to ensure similar loading of samples (Lower).

Fig. 3.

Overlapping binding sites for anti-O-GlcNAc and anti-Ph antibodies on chromosome 3R. Polytene chromosomes were reacted with antibodies to O-GlcNAc (Top) and Ph (Middle). These images were superimposed on chromosomes stained with DAPI, and on each other (Bottom). The locations of the Antennapedia complex (“a”) and bithorax (BX-C) complex (“b”), well-characterized PcG targets, are indicated in Bottom.

Fig. 4.

ChIP analysis of O-GlcNAc modifications at the BX-C locus. (A) A map of the bxd PRE and location of 8 primer pairs used to quantitate binding is shown. (B) Percentage of input binding for each primer pair in embryos is shown. Light gray, IgG control; dark gray, GlcNAc; black, Ph. Negative controls, rp49 (ribosomal protein 49), CG18622 (a gene just outside AbdB of the BX-C that doesn't bind PcG proteins). (C) O-GlcNAc binding is absent in polytene chromosomes of sxc⁻/sxc⁻ mutants. Light gray, IgG; dark gray, GlcNAc WT; black, GlcNAc sxc^−/− mutant.