Transactivation of Fetal Hemoglobin

描述(申请人提供)：镰状细胞病(SCD)是一种常见的遗传病，影响全球数百万人；每年出生的400名非裔美国人中有一人受到影响。羟基脲(HU)等诱导胎儿血红蛋白(HBF)的治疗对患有这种血红蛋白病的患者有巨大的好处，因为持续的-珠蛋白基因表达对这些疾病是缓解的，可能是通过防止红细胞(RBC)的病态和随后的血管闭塞。然而，HU有副作用，长期使用可能致癌。此外，它是过去十年开发的治疗SCD的唯一有效方法。人类类珠蛋白基因转换的发育调控由几个参数控制，主要是反式作用的转录环境和顺式作用的DNA元件。解开控制珠蛋白基因表达的潜在机制，特别是参与激活β-珠蛋白合成的机制，对于识别治疗干预的新靶点是重要的。人类的蛋白质，睾丸特异性蛋白，Y编码的类Y蛋白(TSPYL1)和胎儿珠蛋白诱导因子(FGIF或ANKRD49)，已被证明上调β-珠蛋白基因的表达。这项建议的总体目标是通过确定它们的作用机制来探索这两种蛋白质作为治疗靶点在治疗SCD中的有效性，从而最终可以开发针对这些蛋白质调节的新疗法。对于特定的目标1和2，我们将通过增强TSPYL1和FGIF在红系细胞中的表达(功能获得)或下调表达(功能丧失)来确定体内的表型或发育效应。我们将使用染色质免疫沉淀(ChIP)来确定TSPYL1或FGIF-含复合体结合的3-珠蛋白基因附近的序列，并使用免疫沉淀(IP)结合质谱仪来鉴定TSPYL1或FGIF与之相互作用的配对蛋白。[将评估这两种蛋白质对β-珠蛋白基因激活的特异性。]参与3-珠蛋白基因激活的所有类别的蛋白质都不太可能被发现，留下许多更有益的治疗靶点仍有待发现。因此，对于特定的目的3，我们将采用一种新的选择系统，该系统基于在来自转基因小鼠的？珠蛋白基因座酵母人工染色体(？-YAC)骨髓细胞(？-YAC)中激活A？珠蛋白启动子-绿色荧光蛋白(GFP)融合，以鉴定可能与TSPYL1或FGIF配对的新的3-珠蛋白合成反式激活子。这些研究的完成将为进一步了解TSPYL1和FGIF在发育过程中上调β-珠蛋白基因表达的功能提供重要的动物模型和生化数据。这一建议将产生独特的工具和新的战略，以了解-珠蛋白基因调控治疗SCD的机制。公共卫生相关性：镰状细胞病(SCD)是一种常见的遗传病，影响全球数百万人。每年出生的500名非裔美国人中，有一人会受到SCD的影响。了解控制珠蛋白基因切换的分子机制可能有助于开发靶向治疗或治疗这些疾病，特别是旨在启动胎儿3-珠蛋白基因的研究，这已被证明对SCD的治疗是有效的。

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.