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 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.

Keywords: Ash1; Drosophila; O-GlcNAcylation; O-linked N-acetylglucosamine (O-GlcNAc); RNA polymerase II; SET1; Trithorax; cell cycle; chromatin; polycomb.

FIGURE 1.

O-GlcNAc cycling occurs at sites co-occupied by Polycomb group proteins on promoters of many transcriptionally silenced genes. a, antibodies specific to O-GlcNAc, Pleiohomeotic (Pho), and RNA Pol II Ser2P were used for ChIP-chip analysis. O-GlcNAc and Pho marks overlap significantly at transcriptionally silenced regions. A representative region of the genome, chromosome 3 in the Bithorax cluster, including abd-A and adb-B is shown. Hox genes are occupied by Pho (white) and O-GlcNAc (blue) but lack significant accumulation of RNA Pol II Ser2P (orange, bottom). b, O-GlcNAc levels were detected following OGA RNAi and OGT RNAi in S2 cells. O-GlcNAc levels were increased ∼2-fold following OGA RNAi compared with GFP RNAi. The O-GlcNAc signal was normalized to actin loading control. The graph represents average ± S.D. c, the O-GlcNAc peak intensities of each of ∼8000 regions similar to those shown in a were plotted in increasing order of intensity. Peak intensities increased an average of 1.4-fold in OGA knockdown (red) cells, whereas intensities decreased to near background levels in OGT knockdown (green) cells compared with GFP control RNAi (blue). A 50-gene moving average for OGA and OGT knockdown is highlighted in black, with blue denoting a GFP (WT) control. d, the ratio of peak intensity observed for OGA to WT samples on the Affymetrix Genechip arrays was calculated for each of the roughly 8000 O-GlcNAc occupied chromatin sites. Those intervals that did not change significantly upon OGA knockdown (0.5–1.0) were examined bioinformatically using DAVID functional annotation clustering (42) and found to be 23-fold enriched for Hox genes and other transcriptional regulators (p < 0.0001). Intervals exhibiting an intermediate response to OGA knockdown were 10–12-fold enriched in genes associated with cell cycle and ATP utilization (p < 0.0001). Those showing the greatest change in intensity (1.5–2.5) between OGA and wild type showed a more modest 2–3-fold enrichment for genes associated with ubiquitin and steroid hormone response (p < 0.001).

FIGURE 2.

oga deletion mutant flies have increased protein O-GlcNAcylation. a, the P element that is located upstream of the oga gene was excised by P element excision. The excision removed 657 bp into oga removing the transcription start site and a portion of the second exon. b, the deletion removed the N-terminal 171 amino acids into the predicted O-GlcNAcase domain of the protein. c, oga^del.1 males or females both display an increase in the amount of O-GlcNAc-modified proteins compared with WT or heterozygotes. O-GlcNAc (RL2) and actin are loading controls. Comparison of O-GlcNAc levels after normalization to actin loading control levels in the bar graph shows that oga deletion mutants have ∼6-fold more O-GlcNAc levels than WT. UAS-driven OGA RNAi showed a more modest silencing of OGA as evidenced by the partial increase in O-GlcNAc levels (right panels). d, ectopic expression of OGA (UAS-OGA) with actin-GAL4 promoter restores O-GlcNAc levels similar to WT levels in oga^del.1 mutant background. M, males; F, females. e, oga^del.1 ovaries display diminished O-GlcNAcase activity measured by fluorogenic OGA enzymatic assay. Data represent mean ± S.E. (****, p < 0.0001).

FIGURE 3.

O-GlcNAc levels and number of detected bands increased in oga^del.1 mutant polytene chromosomes. Polytene chromosomes were prepared from WT (w¹¹¹⁸), oga (oga^del.1), and ogt (sxc¹/sxc⁷) mutants. O-GlcNAc signal level (red) and number of bands increased in oga^del.1 mutants. ogt (sxc¹/sxc⁷) mutant polytene is used as negative control for O-GlcNAc staining. Bottom panel shows the enlarged images of dashed boxes in the upper panel for a closer look at the banding pattern on polytenes.

FIGURE 4.

Blocked O-GlcNAc cycling alters the distribution of O-GlcNAc sites with respect to elongating RNA Pol II and sites of Pc repression. Polytene chromosomes were prepared from WT (w¹¹¹⁸) and oga (oga^del.1) mutant flies. The chromosomes were stained with both anti-O-GlcNAc antibody and RNA Pol II Ser2P (elongating RNA Pol II) or Pc. The number of O-GlcNAc and Pol II Ser2P co-localized bands increased in oga^del.1 (left panel, w¹¹¹⁸ versus oga^del.1). O-GlcNAc and Pc shows co-localized polytenes (w¹¹¹⁸, right panel). Many more O-GlcNAc-specific bands were separate from Pc bands (right panel, w¹¹¹⁸ versus oga^del.1). (Select co-localized bands are shown with arrowheads. Lower panel is a blow up of the dashed squares to clearly define bands on the polytenes. O-GlcNAc sites are distinct from RNA Pol II Ser2P, but largely coincident with Pc on polytene chromosomes. Increased O-GlcNAc sites resulting from the oga^del.1 mutant show increased coincidence with RNA Pol II Ser2P and appear at sites distinct from Pc. RNA Pol II Ser2P, and Pc (green), O-GlcNAc (red) and DAPI (blue) are shown. Co-stained bands appear as yellow.

FIGURE 5.

Increased co-localization of ASH1, SET1, and TRX with O-GlcNAc on oga^del.1 mutant polytene chromosomes compared with wild type. Polytene chromosomes prepared from WT (w¹¹¹⁸) and oga mutant (oga^del.1) animals were co-stained for TRX, ASH1, or SET1 along with O-GlcNAc. More bands corresponding to each of these proteins were observed on polytenes of oga^del.1 compared with w¹¹¹⁸ (green signal for each protein). A subset of ASH1, TRX, of SET1 positive bands were also stained with O-GlcNAc (orange-yellow). ASH1, TRX, or SET1, (green), O-GlcNAc (red), and DAPI (blue) are shown. Bottom panel is a blow-up of the marked squares to show bands and co-staining more clearly. Representative co-stained (yellow, orange) bands are shown with arrows.

FIGURE 6.

ASH1, SET1, and TRX are O-GlcNAc modified in oga^del.1 ovaries. Equal amounts of ovary protein extracts were used for immunoprecipitation with TRX, ASH1, SET1, or BRE1 antibodies and run on SDS-PAGE gel. Membranes were immunoblotted for RL2 anti-O-GlcNAc antibody. ASH1 (∼246 kDa), SET1 (∼188 kDa), and TRX (∼400 kDa) are detected by anti-O-GlcNAc antibody (RL2) in oga^del.1 ovary extracts compared with WT. Bre1 (∼115 kDa) was not O-GlcNAc enriched in oga mutants under the same IP conditions. O-GlcNAc and the TRX blot using 10% extract for IPs are shown. Predicted molecular weight of each protein is indicated with an asterisk.

FIGURE 7.

H3 modifications were not altered in oga^del.1 ovaries compared with WT. a, histone 3 modifications of WT and oga^del.1 ovary extracts were analyzed by immunoblotting. No difference in signal was observed for the 9 marks analyzed. b, cell-specific changes in histone marks were examined by generating oga^del.1 germline mosaics. Schematic representation of the generation of mosaics. c, H3K4me, H3K4me3, H3K36me3, and H3K27me3 histone marks were compared between the oga^del.1 mutant (GFP negative) versus neighboring WT or heterozygote cells (GFP positive) in ovary by immunofluorescence. Representative images were stained for GFP (green), DAPI (blue), and H3K4me (red) in ovary germline stem cells (left panel, dashed lines) and H3K4me3 (red) in somatic cells (right panel). WT cells are denoted by an arrow and oga^del.1 cells are denoted by an arrowhead. Signals for each histone mark was normalized to DAPI for each cell; the normalized signal from oga^del.1 cells were then compared with the normalized signal of neighboring WT cells and plotted as fold-change compared with WT (d). Germline stem cells (left graph) and somatic follicular cells (right graph) showed no statistical difference between oga^del.1 and neighboring WT or heterozygote cells. Data represent mean ± S.E.

FIGURE 8.

The expression of cell cycle-related genes HCF and embargoed are increased in oga^del.1 ovaries. The expression of oogenesis and cell cycle-related genes in 5-day-old female ovaries was measured by RT-PCR. Expression of the each gene was normalized to ribosomal RNA. Gene expression of oga^del.1 mutants were then compared with WT and plotted as fold-change to analyze the difference. Cell cycle-related HCF and embargoed showed a nearly 2-fold increase in oga^del.1 mutants, whereas there was no difference in the expression of oogenesis-related genes between oga^del.1 and WT ovaries. Error bars represent mean ± S.E. (*, p < 0.05; **, p < 0.0005).