Transactivation of Fetal Hemoglobin

描述（由申请人提供）：镰状细胞病（SCD）是一种常见的遗传性疾病，影响全球数百万人;它影响每年出生的400名非洲裔美国人中的一名。治疗，如诱导胎儿血红蛋白（HbF）的羟基脲（HU），对患有这种血红蛋白病的患者有巨大的益处，因为持续表达的？珠蛋白基因可能通过防止红细胞（RBC）镰状化和随后的血管闭塞来缓解这些疾病。然而，HU具有负面副作用，长期使用可能致癌。此外，它是过去十年中开发的唯一有效的SCD治疗方法。人类的发展规律？类似珠蛋白的基因转换由几个参数控制，主要是反式作用的转录环境和顺式作用的DNA元件。揭示了珠蛋白基因表达的潜在控制机制，特别是那些参与激活？珠蛋白合成对于识别治疗干预的新靶点是重要的。人类蛋白质，睾丸特异性蛋白质，Y-编码样（TSPYL 1）和胎儿珠蛋白诱导因子（FGIF或ANKRD 49），已被证明上调？珠蛋白基因表达该提案的总体目标是通过确定这两种蛋白质的作用机制来探索它们作为治疗SCD的治疗靶点的有用性，从而最终可以开发针对这些蛋白质的调节的新型疗法。对于特异性目的1和2，我们将在红系细胞中使用TSPYL 1和FGIF的强制表达（功能获得）或表达敲减（功能丧失），以确定体内表型或发育效应。我们将采用染色质免疫沉淀（ChIP），以确定附近的3-珠蛋白基因的序列TSPYL 1或FGIF含有复合物结合，并确定伴侣蛋白TSPYL 1或FGIF相互作用使用免疫沉淀（IP）结合质谱。[The这两种蛋白质的特异性将评估珠蛋白基因激活。3-珠蛋白基因激活中涉及的所有类型的蛋白质都被发现是不可能的，留下许多更有益的治疗靶点仍有待发现。因此，对于特定目标3，我们将采用一种基于A？-珠蛋白启动子-绿色荧光蛋白（GFP）融合蛋白珠蛋白基因座酵母人工染色体（？YAC）骨髓细胞（BMCs），以鉴定可能与或可能不与TSPYL 1或FGIF配对的β-珠蛋白合成的新反式激活因子。这些研究的完成将为进一步了解TSPYL 1和FGIF在上调？珠蛋白基因在发育过程中的表达。这项建议将产生独特的工具和新的战略，以了解机制？用于治疗SCD的珠蛋白基因调控。公共卫生相关性：镰状细胞病（SCD）是一种常见的遗传性疾病，影响全球数百万人。SCD影响每年出生的500名非洲裔美国人中的一人。了解控制珠蛋白基因转换的分子机制可能有助于开发治疗这些疾病的靶向疗法或疗法，特别是旨在开启胎儿3-珠蛋白基因的研究，这已被证明对治疗SCD有效。

DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is a common genetic disease that affects millions of people worldwide; it impacts one of 400 African-Americans born each year. Treatments, such as hydroxyurea (HU), that induce fetal hemoglobin (HbF), have enormous benefit to patients suffering from this hemoglobinopathy, since sustained expression of the ?-globin genes is palliative to these diseases, likely by preventing red blood cell (RBC) sickling and subsequent occlusion of blood vessels. However, HU has negative side effects and may be carcinogenic with long-term use. In addition, it stands alone as the only effective treatment for SCD developed in the last decade. Developmental regulation of human ?-like globin gene switching is controlled by several parameters, primarily the trans-acting transcriptional milieu and cis-acting DNA elements. Unraveling the mechanisms underlying control of globin gene expression, particularly those involved in activation of ?- globin synthesis is important for discerning new targets for therapeutic intervention. The human proteins, testis-specific protein, Y-encoded-like (TSPYL1) and fetal globin inducing factor (FGIF or ANKRD49), have been shown to up-regulate ?-globin gene expression. The overall goal of this proposal is to explore the usefulness of these two proteins as therapeutic targets in treating SCD by determining their mechanisms of action, so that ultimately novel therapies can be developed that target the regulation of these proteins. For Specific Aims 1 and 2, we will use enforced expression (gain-of-function) or knockdown of expression (loss-of function) of TSPYL1 and FGIF in erythroid cells to ascertain phenotypic or developmental effects in vivo. We will employ chromatin immunoprecipitation (ChIP) to determine the sequences near the 3-globin genes where TSPYL1- or FGIF-containing complexes bind, and identify the partner proteins that TSPYL1 or FGIF interact with using immunoprecipitation (IP) coupled with mass spectrometry. [The specificity of these two proteins for ?-globin gene activation will be assessed.] It is improbable that all classes of proteins involved in 3-globin gene activation have been uncovered, leaving many more beneficial therapeutic targets still to be discovered. Thus, for Specific Aim 3, we will employ a novel selection system based on activation of an A?-globin promoter- green fluorescent protein (GFP) fusion in ?-globin locus yeast artificial chromosome (?-YAC) bone marrow cells (BMCs) derived from transgenic mice to identify new transactivators of 3-globin synthesis that may or may not partner with TSPYL1 or FGIF. Completion of these studies will provide important animal models and biochemical data to further understand the function of TSPYL1 and FGIF in up-regulating ?-globin gene expression during development. This proposal will generate unique tools and new strategies to understand mechanisms of ?-globin gene regulation for treatment of SCD. PUBLIC HEALTH RELEVANCE: Sickle cell disease (SCD) is a common genetic disease that affects millions of people worldwide. SCD impacts one of 500 African Americans born each year. Understanding the molecular mechanisms controlling globin gene switching may aid in the development of targeted therapies or therapeutics to treat these diseases, particularly research aimed at turning on the fetal 3-globin genes, which has been shown to be effective for the treatment of SCD.