Drosophila Oga deletion perturbs O-GlcNAcylation of chromatin factors

Drosophila development is a complex and dynamic process regulated, in part, by members of 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 N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), 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. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only

果蝇发育是一个复杂且动态的过程，其调控部分依赖于多梳蛋白（Polycomb, Pc）、三胸蛋白（Trithorax, Trx）以及COMPASS复合物（Compass）这类染色质修饰复合物成员。O-连接N-乙酰葡糖胺转移酶（O-GlcNAc Transferase, OGT/SXC）对Pc介导的基因沉默至关重要，这表明蛋白质的O-连接N-乙酰葡糖胺修饰在发育调控中发挥关键作用。OGT以营养来源的尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）为底物，将N-乙酰-D-葡糖胺（N-acetyl-D-glucosamine, GlcNAc）转移至关键转录调控因子的丝氨酸或苏氨酸残基的羟基上，而该修饰可被O-葡糖胺酶（O-GlcNAcase, OGA）动态移除。我们在果蝇S2细胞中对OGT或OGA进行RNA干扰敲低后开展了染色质免疫沉淀-芯片（ChIP-chip）与微阵列分析，结果发现，在OGA敲低的细胞中，全基因组范围内的O-连接N-乙酰葡糖胺修饰水平显著升高，尤其富集于有丝分裂与细胞周期相关基因区域，但并未在多梳蛋白家族成员Pleiohomeotic（Pho）共同结合的位点（如同源框Hox与NK同源框基因簇）处出现该现象。微阵列分析结果显示，O-连接N-乙酰葡糖胺循环失衡会扰乱与形态发生及细胞周期调控相关基因的表达。为探究整体动物体内O-连接N-乙酰葡糖胺循环紊乱的体内效应，我们在果蝇中构建了oga的无效等位基因（oga^del.1）。在oga^del.1突变体中，包括三胸蛋白家族成员Trx、Absent small or homeotic discs 1（Ash1）以及COMPASS复合物成员Set1组蛋白甲基转移酶在内的表观遗传激活因子均发生了O-连接N-乙酰葡糖胺修饰。oga^del.1突变体的卵巢中，一组独特的细胞周期相关基因的表达模式发生改变。我们的研究结果表明，在果蝇中，OGA的缺失会通过在众多染色质调控因子（包括Trx、Ash1与Set1）上积累O-连接N-乙酰葡糖胺修饰，进而影响表观遗传调控系统。我们在果蝇S2细胞中对OGT或OGA进行RNA干扰敲低后开展了Affymetrix瓦片阵列分析，以验证OGA敲低细胞中全基因组O-连接N-乙酰葡糖胺修饰水平升高的现象，该现象尤其富集于有丝分裂与细胞周期相关基因区域，而未出现在Pc家族成员Pho共同结合的位点——此部分仅代表基因表达组学数据。