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)与其他血红蛋白疾病(α-和β-地中海贫血)一起是人类最常见的遗传性疾病，通常患者需要终生接受治疗。目前的治疗方式要么有令人衰弱的副作用，要么对主要的受影响个体无效。仅在美国，SCD患者每年的医疗费用就超过11亿美元/年(约2008年)。临床和实验研究都发现，即使γ-珠蛋白表达略有增加，也可以缓解SCD的症状、器官病理和疼痛。我们的实验室发现了一个与ε和γ珠蛋白基因抑制有关的大分子阻遏复合体，该复合体的核心成分是孤儿核受体Tr2(NR2C1)和Tr4(NR2C2)。当抑制物复合体被抑制时，γ-珠蛋白的表达增加，SCD的病理生理表现减少，从而使其成为有吸引力的治疗开发的靶点。然而，除了ε-和γ-珠蛋白基因外，所有二聚体受体物种(即Tr2同源二聚体、Tr4同源二聚体或Tr2/Tr4异二聚体)的转录靶标和全基因组结合，以及每个物种激活或抑制红系转录的作用(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。我们的主要假设是，在红系分化过程中，TR2和TR4同源和异源二聚体形成不同的转录复合体，激活或抑制靶基因的转录。特定目标1将通过CHIP-SEQ方法在阶段纯化的小鼠红系祖细胞中检测TR2和TR4同源二聚体和异源二聚体的直接基因组结合部位和转录靶点，并利用融合二聚体来检测特定的二聚体活性。在特定的目标2中，我们将研究红细胞中TR2和TR4同源二聚体和异源二聚体复合体与DNA的结合机制。该项目的发现将使我们更深入地了解TR2和TR4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新的治疗方法，与目前用于治疗血红蛋白疾病的药物相比，这些药物具有更强的特异性和更少的副作用。

 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.