RESUMEN
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.
Asunto(s)
Acetilglucosamina/metabolismo , Cromatina/metabolismo , Drosophila/enzimología , Epigénesis Genética/genética , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional , Eliminación de Secuencia , Animales , Western Blotting , Células Cultivadas , Cromatina/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Inmunoprecipitación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TranscriptomaRESUMEN
O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear. Here, we report a conditional disruption of the Oga gene in the mouse. The resulting homozygous Oga null (KO) animals lack OGA enzymatic activity and exhibit elevated levels of the O-GlcNAc modification. The Oga KO animals showed nearly complete perinatal lethality associated with low circulating glucose and low liver glycogen stores. Defective insulin-responsive GSK3ß phosphorylation was observed in both heterozygous (HET) and KO Oga animals. Although Oga HET animals were viable, they exhibited alterations in both transcription and metabolism. Transcriptome analysis using mouse embryonic fibroblasts revealed deregulation in the transcripts of both HET and KO animals specifically in genes associated with metabolism and growth. Additionally, metabolic profiling showed increased fat accumulation in HET and KO animals compared with WT, which was increased by a high fat diet. Reduced insulin sensitivity, glucose tolerance, and hyperleptinemia were also observed in HET and KO female mice. Notably, the respiratory exchange ratio of the HET animals was higher than that observed in WT animals, indicating the preferential utilization of glucose as an energy source. These results suggest that the loss of mouse OGA leads to defects in metabolic homeostasis culminating in obesity and insulin resistance.
Asunto(s)
Técnicas de Inactivación de Genes , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Prueba de Tolerancia a la Glucosa , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Homeostasis , Humanos , Insulina/metabolismo , Ratones , Ratones Noqueados , Muerte Perinatal/etiología , Periodo Posparto , EmbarazoRESUMEN
Proper cellular functioning requires that cellular machinery behave in a spatiotemporally regulated manner in response to global changes in nutrient availability. Mounting evidence suggests that one way this is achieved is through the establishment of physically defined gradients of O-GlcNAcylation (O-linked addition of N-acetylglucosamine to serine and threonine residues) and O-GlcNAc turnover. Because O-GlcNAcylation levels are dependent on the nutrient-responsive hexosamine signaling pathway, this modification is uniquely poised to inform upon the nutritive state of an organism. The enzymes responsible for O-GlcNAc addition and removal are encoded by a single pair of genes: both the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA, also known as MGEA5) genes are alternatively spliced, producing protein variants that are targeted to discrete cellular locations where they must selectively recognize hundreds of protein substrates. Recent reports suggest that in addition to their catalytic functions, OGT and OGA use their multifunctional domains to anchor O-GlcNAc cycling to discrete intracellular sites, thus allowing them to establish gradients of deacetylase, kinase and phosphatase signaling activities. The localized signaling gradients established by targeted O-GlcNAc cycling influence many important cellular processes, including lipid droplet remodeling, mitochondrial functioning, epigenetic control of gene expression and proteostasis. As such, the tethering of the enzymes of O-GlcNAc cycling appears to play a role in ensuring proper spatiotemporal responses to global alterations in nutrient supply.
Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/metabolismo , Animales , Humanos , Procesamiento Proteico-Postraduccional , Transducción de Señal/fisiologíaRESUMEN
Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.