Mode of Action of GCM2, the Essential Parathyroid Transcription Factor

描述（由申请人提供）：关于GCM 2（必需的甲状旁腺转录因子）的特定作用机制，已知的信息量少得令人惊讶;这些信息对于鉴定甲状旁腺疾病的新的潜在药物靶点至关重要。甲状旁腺的功能-产生甲状旁腺激素（PTH）-是维持正常钙稳态和骨骼健康所必需的。甲状旁腺疾病代表了一些最常见的内分泌疾病，包括原发或继发于慢性肾脏疾病的甲状旁腺功能亢进。导致甲状旁腺功能减退的GCM 2突变和GCM 2-/-小鼠的结果表明，GCM 2是甲状旁腺发育的主要调节因子。它不仅在胚胎发育期间表达，而且在成熟器官中也表达。这种独特的转录因子如何指导和维持甲状旁腺特异性程序？传统方法无法解决这个问题。理解这一机制的最大障碍是缺乏可用的甲状旁腺细胞系和缺乏研究蛋白质-蛋白质相互作用所需的有效GCM 2抗体。为了克服这些障碍，我们建立了一种新的基因敲入小鼠模型，携带双标签GCM 2，它保留了GCM 2的正常行为。这种独特的体内工具现在将使靶基因和细胞特异性结合伴侣的鉴定成为可能。我们的长期目标是靶向GCM 2转录网络的组成部分，以治疗甲状旁腺疾病。在我们的初步数据的支持下，我们的中心假设是GCM 2通过调节细胞特异性靶基因网络的启动子和增强子以及与特定蛋白质伴侣结合来起作用。本提案的目的是系统地确定GCM 2及其结合伙伴的靶基因，并进一步表征这些组分的作用机制。为了实现这些目标，我们特别提出（1）使用来自我们的基因敲入小鼠模型的甲状旁腺组织确定甲状旁腺中GCM 2的DNA结合位点和靶基因。我们将使用ChIP-seq和DNase-seq技术，然后对识别的靶基因进行初步功能分析。此外，我们将（2）使用免疫共沉淀和串联质谱法鉴定GCM 2的结合伴侣，并对其进行初步功能表征。这项研究的贡献将是重大的，因为这种关键的甲状旁腺调节剂的作用目前是一个“黑匣子”-靶基因和其他球员大多是未知的。在本申请中建议的方法，即使用表位标记的敲入小鼠模型，是创新的，因为它关注于一种新的方法，使用表位标记的敲入模型而不是缓慢且低效的现有候选方法。基于GCM 2对甲状旁腺的核心作用，我们的研究结果预计将产生重要的积极影响;除了推进甲状旁腺生物学领域外，所确定的组分极有可能为治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): A surprisingly small amount of information is known about the specific mechanism of action of GCM2, the essential parathyroid transcription factor; information that is critical for identifying new, potential drug targets for parathyroid gland diseases. The function of the parathyroid glands-to produce parathyroid hormone (PTH)-is essential for maintaining normal calcium homeostasis and bone health. Diseases of the parathyroid glands represent some of the most common endocrine disorders, including hyperparathyroidism, which is primary or secondary to chronic kidney disease. Mutations in GCM2 that cause hypoparathyroidism, and the results of GCM2-/- mice, reveal that GCM2 is a master regulator of parathyroid gland development. It is expressed not only during embryonic development, but also in the mature organ. How does this unique transcription factor direct and maintain the parathyroid gland-specific program? Traditional methods have not been able to address this question. The largest hurdles to understanding this mechanism have been the lack of an available parathyroid cell line and the absence of potent GCM2 antibodies needed to study protein-protein interactions. To circumvent these obstacles, we have established a novel knock-in mouse model carrying a double-tagged GCM2, which preserves the normal behavior of GCM2. This unique in vivo tool will now make possible the identification of target genes and cell-specific binding partners. Our long-term goal is to target components of the GCM2 transcriptional network in order to treat diseases of the parathyroid glands. Supported by our preliminary data, our central hypothesis is that GCM2 works through regulating promoters and enhancers of a network of cell-specific target genes and by binding to specific protein partners. The objectives of this proposal are to systematically identify the target genes of GCM2 and its binding partners and to further characterize the mechanism of action of these components. To approach these goals, we specifically propose to (1) determine DNA binding sites and target genes for GCM2 in the parathyroid gland using parathyroid tissue from our knock-in mouse model. We will be using ChIP-seq and DNase-seq techniques followed by the initial functional analysis of identified target genes. Furthermore, we will (2) identify binding partners of GCM2 using co-immunoprecipitation and tandem mass spectrometry, and perform their initial functional characterization. The contribution of this research will be significant because the action of this key parathyroid regulator is currently a "black box" - target genes and other players are mostly unknown. The approach suggested in this application, namely the use of an epitope-tagged knock-in mouse model, is innovative, because it focuses on a novel approach, using an epitope-tagged knock-in model instead of the slow and inefficient existing candidate approach. Based on the central role of GCM2 for the parathyroid glands, our results are expected to have an important positive impact; in addition to advancing the field of parathyroid gland biology, the identified components are highly likely to provide new targets for therapeutic interventions.