Lnc'ing White Fat to Brown Fat Thermogenesis

摘要 大约有1.6亿美国男性、女性和儿童超重或肥胖。几乎75%的男性， 60%的女性和30%的20岁以下的男孩和女孩肥胖或超重。肥胖与 与包括糖尿病、心血管疾病和癌症在内的多种合并症。有两种类型的 胖了棕色脂肪在新生儿和小型哺乳动物中很常见。它是有用的产生热量，特别是在小 容易散热的物体白色脂肪随着年龄的增长而增加，用于储存能量和绝缘 防止热量流失。增加的白色脂肪与增加的肥胖相关疾病有关，而增加的 棕色脂肪与减少肥胖相关疾病有关。 最近几项关于脂肪组织的研究已经确定了与棕色脂肪相关的转录特征 脂肪生成和特别是产热程序。其中一些研究还发现了长期的非- 编码与白色脂肪和棕色脂肪特性和功能相关的RNA（lncRNA）。但 这些lncRNA如何影响转录程序并决定棕色脂肪身份的机制， 功能知之甚少。 我们建议使用我们最近开发的基于lncRNA的酵母三杂交（Y3 H）试验， 定义了318种与棕色脂肪脂肪形成有关的保守lncRNA的lncRNA-蛋白质相互作用。我们的Y3 H 系统能够检测lncRNA-蛋白质相互作用，否则由于以下问题而难以检测lncRNA-蛋白质相互作用： 低丰度和低亲和力的相互作用。将lncRNA-蛋白质相互作用叠加到现有蛋白质上- 蛋白质相互作用网络将定义正常棕色脂肪脂肪形成的lncRNA调节途径， 还鉴定了可能被操纵以增加产热的lncRNA调节途径。因为我们 将筛选这318个单独lncRNA的重叠lncRNA片段，这些研究将使 系统lncRNA结构-功能分析通过这种结构-功能获得的信息 分析对于建立序列和/或结构参数是至关重要的，这些参数可以预测 lncRNA-蛋白结合活性，从而推进我们对lncRNA生物学的理解，即使是那些 不包括在这个初始屏幕中。 对于一组lncRNA-蛋白质相互作用，我们将通过以下方法验证其在棕色脂肪脂肪形成小鼠模型中的作用： 增加（通过异位表达）或减少（通过敲低）lncRNA及其相关蛋白， 棕色脂肪祖细胞我们将通过测试肥胖、棕色脂肪谱系和肥胖症的发生率来评估这些相互作用的影响。 定型（通过FACS分析）和产热（通过转录谱分析）。这些研究将（1） 鉴定指导脂肪生成和产热的lncRNA-蛋白质相互作用;（2）开发用于 系统研究lncRNA;（3）提供临床前证据支持靶向lncRNA的临床试验- 肥胖相关疾病的直接途径。

ABSTRACT Approximately 160 million American men, women and children are overweight or obese. Almost 75% of men, 60% of women and 30% of boys and girls under the age of 20 are obese or overweight. Obesity is associated with numerous co-morbidities including diabetes, cardiovascular disease, and cancers. There are two types of fat. Brown fat is common in newborns and small mammals. It is useful for generating heat, especially in small bodies that tend to lose heat. White fat increases as people age and is useful for storing energy and insulating against heat loss. Increased white fat is associated with increased obesity-related disease whereas increased brown fat is associated with reduced obesity-related disease. Several recent studies of adipose tissue have identified transcriptional signatures associated with brown fat adipogenesis and specifically thermogenic programs. Some of these studies have also identified long non- coding RNAs (lncRNAs) associated with white fat and brown fat identity and function. However, the mechanism(s) of how these lncRNAs affect transcriptional programs and determine brown fat identity and function are poorly understood. We propose to use our recently developed lncRNA-based yeast three hybrid (Y3H) assay to systematically define lncRNA-protein interactions for 318 conserved lncRNAs implicated in brown fat adipogenesis. Our Y3H system is capable of detecting lncRNA-protein interactions that are otherwise difficult to detect due to issues of low-abundance and low-affinity interactions. Overlaying lncRNA-protein interactions onto existing protein- protein interaction networks will define lncRNA-regulated pathways of normal brown fat adipogenesis and will also identify lncRNA-regulated pathways that might be manipulated to increase thermogenesis. Because we will screen overlapping lncRNA fragments of these 318 individual lncRNAs, these studies will enable systematic lncRNA structure-function analyses. The information obtained through this structure-function analysis will be critical for establishing sequence and/or structural parameters which may enable prediction of lncRNA-protein binding activity, thereby advancing our understanding of the biology of lncRNAs, even those not included in this initial screen. For a set of lncRNA-protein interactions, we will validate roles in mouse models of brown fat adipogenesis by increasing (by ectopic expression) or decreasing (by knockdown) lncRNAs and their associated proteins in brown fat progenitor cells. We will assess the effects of these interactions by testing obesity, brown fat lineage commitment (by FACS analysis) and thermogenesis (by transcriptional profiling). Thus, these studies will (1) identify lncRNA-protein interactions directing adipogenesis and thermogenesis; (2) develop a platform for systemically studying lncRNAs; and (3) provide pre-clinical evidence support clinical trials targeting lncRNA- directed pathways for obesity-related diseases.