Characterization of TR2 and TR4 homodimer and heterodimer transcriptional mechanisms during erythropoiesis.

 描述（由申请人提供）：镰状细胞病（SCD）与其他血红蛋白病（α-地中海贫血和β-地中海贫血）一起，是人类最常见的遗传性疾病，患者通常需要终生治疗。目前的治疗方式要么有使人衰弱的副作用，要么对受影响个体的主要子集无效。仅在美国，SCD患者的年度医疗护理费用就超过11亿美元/年（约100亿美元）。2008年）。临床和实验研究都发现，即使是γ-珠蛋白表达的适度增加也可以减轻SCD中的症状、器官病理学和疼痛。我们的实验室鉴定了一种负责抑制ε-和γ-珠蛋白基因的大分子阻遏物复合物，该复合物的核心DNA结合组分是孤儿核受体TR 2（NR 2C 1）和TR 4（NR 2C 2）。当这种阻遏物复合物被抑制时，γ-珠蛋白的表达增加，SCD的病理生理学表现减少，从而使其成为治疗开发的有吸引力的靶点。然而，除了ε-和γ-珠蛋白基因之外，所有二聚体受体种类（即TR 2同二聚体、TR 4同二聚体或TR 2/TR 4异二聚体）的转录靶点和全基因组结合或每个种类激活或抑制红细胞转录的作用（例如它们与共激活子和共抑制子的差异关联）在很大程度上是未知的。我们的总体假设是，TR 2和TR 4同源和异源二聚体形成不同的转录复合物，在红系分化过程中激活或抑制靶基因转录。具体目标1将在阶段纯化的鼠红系祖细胞中通过ChIP-seq方法检查TR 2和TR 4同源二聚体和异源二聚体的直接基因组结合位点和转录靶标，并利用融合二聚体检查特异性二聚体活性。在具体目标2中，我们将研究指导TR 2和TR 4同源二聚体和异源二聚体复合物与红细胞中DNA结合的机制。该项目的发现将使人们更深入地了解TR 2和TR 4在正常红细胞生成过程中的功能，并更好地了解控制珠蛋白基因表达的分子机制;反过来，这些发现将有助于开发比目前用于治疗血红蛋白病的药物具有更高特异性和更少副作用的新疗法。

 DESCRIPTION (provided by applicant): Sickle cell disease (SCD) when included with other hemoglobinopathies (α- and β-thalassemia) are the most common inherited diseases in man, and most often patients require treatment for their entire lives. Current treatment modalities either have debilitating side effects or are not effective for a major subset of affected individuas. The annual cost of medical care of SCD patients in the US alone exceeds $1.1 billion/annum (ca. 2008). Both clinical and experimental studies have found that even a modest increase in γ-globin expression can alleviate symptoms, organ pathology, and pain in SCD. Our laboratory identified a macromolecular repressor complex responsible for repression of the ε- and γ-globin genes, and the core DNA binding components of this complex are the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2). When this repressor complex is inhibited, the expression of γ-globin increases and the pathophysiological manifestations of SCD were reduced, thus making it an attractive target for therapeutic development. However, other than the ε- and γ-globin gene the transcriptional targets and genome-wide binding of all dimeric receptor species (i.e. TR2 homodimer, TR4 homodimer, or TR2/TR4 heterodimer) or the actions by which each species activates or represses erythroid transcription (e.g. their differential association with co-activatrs and co-repressors) are largely unknown. Our overarching hypothesis is that TR2 and TR4 homo- and heterodimers form distinct transcriptional complexes to either activate or repress target gene transcription during erythroid differentiation. Specific aim 1 will examine the direct genomic binding sites and transcriptional targets of TR2- and TR4- homodimers and heterodimers through a ChIP-seq approach in stage purified murine erythroid progenitor cells, and utilization of fused dimers to examine specific dimer activity. In Specific aim 2, we will examine the mechanisms that direct the binding of TR2 and TR4 homodimers and heterodimer complexes to DNA in erythrocytes. The discoveries from this project will provide a deeper understanding of the functions of TR2 and TR4 during normal erythropoiesis, and provide a better understanding of the molecular mechanisms controlling globin gene expression; in turn, these findings will aid in the development of new therapeutics with greater specificity and fewer negative side effects than current medications used to treat the hemoglobinopathies.