The Role of Myc in ChREBP-dependent, Glucose-stimulated Gene Expression

描述（由申请人提供）：细胞感知和响应其代谢环境变化的能力对于预防代谢综合征和糖尿病的病理生理后果至关重要。细胞对代谢燃料可用性变化的适应包括基因表达模式的协调变化，由营养感测转录因子介导，并且该过程在糖尿病中被破坏，尽管有丰富的代谢燃料，细胞仍被困在禁食状态。本提案的目的是了解两个转录因子参与营养传感和肝脏禁食到进食的过渡，葡萄糖敏感转录因子碳水化合物反应元件结合蛋白（ChREBP）和Myc之间的关系。ChREBP介导代谢相关组织（如肝脏和胰腺β细胞）中葡萄糖响应性靶基因的转录激活。葡萄糖刺激ChREBP易位到细胞核，一旦ChREBP与靶基因结合，则必须发生依赖于葡萄糖的其他定义不明确的分子事件才能进行反式激活。失调的Myc和最近的ChREBP都涉及与癌症转化相关的糖酵解增加（瓦尔堡效应）。我们发表的初步数据表明，Myc是ChREBP依赖性葡萄糖刺激的基因表达所必需的，被招募到葡萄糖响应基因的调控区，并且是招募ChREBP和葡萄糖响应的其他激活转录因子所必需的。我们假设Myc在更大的背景下起作用，以允许代谢表型之间的转换，例如，禁食和进食状态。我们的挑战，反映在这个建议是更完整地了解Myc和ChREBP之间的分子关系，在葡萄糖稳态中发挥的生理作用的背景下，这样我们就可以更好地了解如何在疾病状态下进行治疗。我们的总体假设是Myc是ChREBP依赖性葡萄糖反应和代谢表型之间转换所必需的。具体目标1）是确定消耗肝脏中Myc活性的生理、代谢和分子后果。使用CRE-Lox方法，我们将通过RT-PCR和SOLID SAGE测量基因表达;通过ChIP和ChIP-seq测量ChREBP和其他转录因子;通过测量糖酵解、脂肪生成、脂质储存和氧化、酮生成和葡萄糖输出，以及全身和肝脏胰岛素抵抗和葡萄糖耐量，确定肝脏Myc耗竭对禁食至进食过渡的影响。具体目标2是确定Myc如何促进ChREBP依赖性葡萄糖激活的基因转录。我们将使用蛋白质组学方法来测试葡萄糖是否改变Myc的翻译后修饰;我们将确定Myc是否以葡萄糖依赖性方式改变葡萄糖应答基因的表观遗传环境;并将绘制Myc葡萄糖特异性功能所需的Myc结构域。实现这些目标将为理解Myc和ChREBP之间的正常生理关系提供框架，并将允许代谢紊乱如代谢综合征和糖尿病的治疗的知情设计，并可能很好地提供对癌症代谢扰动的见解。

DESCRIPTION (provided by applicant): The ability of cells to sense and respond to changes in their metabolic environment is crucial to preventing the pathophysiological consequences of metabolic syndrome and diabetes. Cellular adaptation to changes in metabolic fuel availability includes coordinated changes in patterns of gene expression, mediated by nutrient- sensing transcription factors, and this process is disrupted in diabetes, with cells trapped in the fasted state despite abundant metabolic fuel. The purpose of the present proposal is to understand the relationship between two transcription factors involved in nutrient sensing and the hepatic fasted-to-fed transition, the glucose-sensing transcription factor carbohydrate response element binding protein (ChREBP), and Myc. ChREBP mediates transcriptional activation of glucose-responsive target genes in metabolically relevant tissues such as the liver and pancreatic beta cells. Glucose stimulates translocation of ChREBP to the nucleus, and once ChREBP is bound to target genes, additional poorly defined molecular events, dependent on glucose, must take place for transactivation. Dysregulated Myc, and recently ChREBP, have both been implicated in the increased glycolysis (Warburg effect) associated with transformation in cancer. Our published and Preliminary Data demonstrate that Myc is required for ChREBP-dependent glucose-stimulated gene expression, is recruited to regulatory regions of glucose-responsive genes, and is necessary for the recruitment of ChREBP and other activating transcription factors of the glucose response. We hypothesize that Myc works in a larger context to allow transition between metabolic phenotypes, e.g., the fasted and fed states. Our challenge, reflected in this proposal is to understand more completely the molecular relationship between Myc and ChREBP within the context of the physiological roles they play in glucose homeostasis, so that we can better understand how to manipulate them therapeutically in disease states. Our overarching hypothesis is that Myc is required for the ChREBP-dependent glucose response, and for transitions between metabolic phenotypes. Specific Aim 1) is to determine the physiological, metabolic, and molecular consequences of depleting Myc activity in the liver. Using a CRE-Lox approach, we will determine the effect of hepatic Myc depletion on the fasting-to-fed transition by measuring gene expression with RT-PCR and SOLID SAGE; by measuring ChREBP and other transcription factor occupancies by ChIP and by ChIP-seq; and by measuring glycolysis, lipogenesis, lipid storage and oxidation, ketogenesis, and glucose output, as well as whole body and hepatic insulin resistance and glucose tolerance. Specific Aim 2 is to determine how Myc promotes ChREBP-dependent glucose-activated gene transcription. We will use a proteomic approach to test if glucose alters the posttranslational modification of Myc; we will determine if Myc alters the epigenetic environment of glucose responsive genes in a glucose-dependent manner; and will map the domains of Myc required for the glucose-specific functions of Myc. Accomplishing these Aims will provide the framework for understanding the normal physiological relationship between Myc and ChREBP, and will allow informed design of therapies for metabolic disorders such as metabolic syndrome and diabetes, and may well provide insights towards the metabolic perturbations of cancer.