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 O-GlcNAcylation 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 after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, we observed a genome-wide increase in the intensity of most O-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. We found 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 of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.
Highlights
Epigenetic regulation of gene expression during development is essential for proper cell fate determination
We found that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in ogadel.1 mutants
Our results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1
Summary
Drosophila S2 Cell Culture and RNAi Treatment—S2 cells were cultured in Schneider’s Drosophila medium supplemented with 10% FBS. Pho, O-GlcNAc, and RNA Pol II Ser2P ChIP-chip was performed by a minor modification of the method described previously [19]. ChIP-on-Chip Analysis—ChIP-chip analysis was carried out in Drosophila S2 cells by a modification of the method described previously using anti-O-GlcNAc antibody (mouse HGAC-85) [19]. The co-enrichment of O-GlcNAc, Pho, RNA Pol II Ser2P, and other chromatin factors were determined using Affymetrix Tiling Analysis Software version 1.1. After 3 washes with PBS/Triton X-100, the slides were blocked with Odyssey blocking reagent for 1 h at room temperature, and incubated with ASH1, TRX, or SET1 at 1/50 dilution along with O-GlcNAc specific antibody at 1/100 dilution overnight at 4 °C in a humidified chamber.
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