Transactivation of Fetal Hemoglobin

描述（由申请人提供）：镰状细胞病（SCD）是一种常见的遗传病，影响全世界数百万人；它影响每年出生的 400 名非裔美国人中的一名。诱导胎儿血红蛋白 (HbF) 的羟基脲 (HU) 等治疗方法对患有这种血红蛋白病的患者具有巨大益处，因为 β-珠蛋白基因的持续表达可以缓解这些疾病，可能是通过防止红细胞 (RBC) 镰状化和随后的血管闭塞。然而，HU有副作用，长期使用可能致癌。此外，它是过去十年中开发的唯一有效的 SCD 治疗方法。人类β样珠蛋白基因转换的发育调控由几个参数控制，主要是反式作用转录环境和顺式作用DNA元件。揭示珠蛋白基因表达控制的机制，特别是那些参与β-珠蛋白合成激活的机制，对于识别治疗干预的新靶点非常重要。人类蛋白质、睾丸特异性蛋白质、Y 编码样蛋白 (TSPYL1) 和胎儿珠蛋白诱导因子（FGIF 或 ANKRD49）已被证明可以上调 β-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的作用机制来探索这两种蛋白作为治疗靶点在治疗 SCD 中的有用性，以便最终开发出针对这些蛋白调节的新疗法。对于具体目标 1 和 2，我们将使用 TSPYL1 和 FGIF 在红系细胞中的强制表达（功能获得）或表达敲低（功能丧失）来确定体内表型或发育效应。我们将采用染色质免疫沉淀 (ChIP) 来确定包含 TSPYL1 或 FGIF 的复合物结合的 3-珠蛋白基因附近的序列，并使用免疫沉淀 (IP) 结合质谱来鉴定 TSPYL1 或 FGIF 相互作用的伴侣蛋白。 [将评估这两种蛋白质对 β-珠蛋白基因激活的特异性。] 不可能发现参与 3-珠蛋白基因激活的所有蛋白质类别，因此仍有许多更有益的治疗靶点有待发现。因此，对于特定目标 3，我们将采用基于源自转基因小鼠的 β-珠蛋白位点酵母人工染色体 (β-YAC) 骨髓细胞 (BMC) 中 Aβ-珠蛋白启动子-绿色荧光蛋白 (GFP) 融合物激活的新型选择系统，以鉴定可能与 TSPYL1 或 FGIF 合作或不合作的新 3-珠蛋白合成反式激活因子。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。该提案将产生独特的工具和新策略来了解用于治疗 SCD 的 β-珠蛋白基因调控机制。公共卫生相关性：镰状细胞病 (SCD) 是一种常见的遗传病，影响全世界数百万人。 SCD 影响每年出生的 500 名非裔美国人中的一名。了解控制珠蛋白基因转换的分子机制可能有助于开发靶向疗法或治疗这些疾病的疗法，特别是旨在开启胎儿 3-珠蛋白基因的研究，该基因已被证明可有效治疗 SCD。

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.