Drosophila O-GlcNAcase deletion globally perturbs chromatin O-GlcNAcylation

Abstract

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/ SXC) is essential for Pc repression suggesting that the OGlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, we performed ChIP-chip and microarray analysis afterOGTorOGARNAi knockdown in S2 cells. AfterOGA RNAi, we observed a genome-wide increase in the intensity of mostO-GlcNAc-occupied regions including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. We then produced a viable null allele of oga (oga del.1 ) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. Wefound that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga del.1 mutants. The oga del.1 mutants displayed altered expression of a distinct set of cell cycle-related genes. Our results show that the loss ofOGAin Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.