Nutrient Flux and Development

养分流动与发育

• 批准号: 7967365
• 负责人: Michael Krause
• 金额: $ 49.56万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967365
• 关键词: Acetylglucosamine; Affect; Aging; Alleles; Animal Model; Animals; Antibodies; Area; Behavior; Biological Assay; C. elegans genome; Caenorhabditis elegans; Cells; Chromatin; Collaborations; Defect; Dense Core Vesicle; Development; Diabetes Mellitus; Disease; Enzymes; Excision; Gene Expression; Gene Expression Regulation; Genes; Genetic Models; Genomics; Hexosamines; Homeostasis; Human; Insulin; Knock-out; Link; Longevity; Mediating; Membrane Proteins; Metabolic Diseases; Metabolism; Mutation; National Institute of Dental and Craniofacial Research; National Institute of Diabetes and Digestive and Kidney Diseases; Nematoda; Neurosecretion; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Pore; Nutrient; Nutritional; Pathway interactions; Pattern; Phosphotransferases; Physiology; Play; Pore Proteins; Proteins; RNA; Regulation; Reporter; Role; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Testing; Tissues; Toxic effect; Transcriptional Regulation; Work; chromatin immunoprecipitation; detection of nutrient; follow-up; in vivo; insight; insulin secretion; man; mutant; novel; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; prevent; promoter; response; transcription factor

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease. In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized a knockout alleles of ogt-1 and oga-1 genes. Using a combination of genomic expression arrays and chromatin immunoprecipitation (ChIP) we are looking for genes that respond to nutrient flux differently in the mutants with the hope of identifying pathways of importance. The expression analysis has revealed widespread de-regulation of gene expression in the mutants, identify affected pathways including longevity and aging. We have tested these pathways in the mutants and find alteration in function that are consistent with the gene expression patterns we observe. From the ChIP studies, we have identified a discrete number of genes associated with O-GlcNAcylated proteins. These associations are pronounced at the promoters of the genes and show some overlap with ChIP signals using RNA PolII antibodies. We are currently investigating the function role, if any, of these restricted O-GlcNAc chromatin marks. These marks have the potential to link nutritional flux in the cell directly to gene regulation, offering a novel insight into the role of O-GlcNAc cycling in animal physiology and development. In collaboration with the Notkins lab (NIDCR) we have followed up on our earlier studies of a protein called IA-2. This membrane protein associated with dense core vesicles plays an important role in neurosecretion from a variety of tissues. Using an in vivo reporter for IA-2, we have screened for C. elegans mutants that can not properly control IA-2 levels. One such mutant turned out to be in the gene called pag-3 encoding a transcription factor. Our studies have demonstrated that mis-regulation of IA-2 in pag-3 mutants results in a disruption of dense core vesicle synthesis or stability resulting in neurosecretory defects. Our work demonstrated a novel layer of transcriptional regulation impacting dense core vesicle dynamics with implications for diseases of neurosecretion, including diabetes.

O-连接的N-乙酰葡糖胺（O-GlcNAc）添加和去除的动态循环作用于核孔蛋白、转录因子和激酶，以调节细胞信号级联。这种营养传感己糖胺信号通路是保守的从线虫到man.A单核苷酸多态性在人类O-GlcNAcase基因与2型糖尿病，这表明该通路的扰动导致疾病。 在与汉诺威实验室（NIDDK）的合作中，我们证明了C。线虫基因组编码两种进化上保守的酶，其介导O-GlcNAc循环，基因称为ogt-1和oga-1。 我们以前的特点是敲除等位基因ogt-1和oga-1基因。使用基因组表达阵列和染色质免疫沉淀（ChIP）的组合，我们正在寻找基因，响应不同的营养通量的突变体，希望确定的重要途径。表达分析揭示了突变体中广泛的基因表达失调，确定了受影响的途径，包括长寿和衰老。 我们已经在突变体中测试了这些途径，并发现与我们观察到的基因表达模式一致的功能改变。 从ChIP研究中，我们已经鉴定了与O-GlcNAc酰化蛋白相关的离散数量的基因。 这些关联在基因的启动子处是明显的，并且使用RNA PolII抗体显示出与ChIP信号的一些重叠。 我们目前正在研究这些限制性O-GlcNAc染色质标记的功能作用（如果有的话）。 这些标记有可能将细胞中的营养通量直接与基因调控联系起来，为O-GlcNAc循环在动物生理学和发育中的作用提供了新的见解。 在与Notkins实验室（NIDCR）的合作中，我们跟进了我们早期对IA-2蛋白质的研究。 这种与致密核心囊泡相关的膜蛋白在多种组织的神经分泌中起重要作用。 使用IA-2的体内报告基因，我们筛选了C。elegans突变体不能正确控制IA-2水平。 其中一个突变体被证明存在于编码转录因子的pag-3基因中。 我们的研究表明，在pag-3突变体中IA-2的错误调节导致致密核心囊泡合成或稳定性的破坏，从而导致神经分泌缺陷。 我们的工作证明了一层新的转录调节，影响致密核心囊泡动力学，对包括糖尿病在内的神经分泌疾病具有影响。