Transactivation of Fetal Hemoglobin

镰状细胞病（SCD）是一种常见的遗传性疾病，影响全世界数百万人;它 影响了每年出生的400名非裔美国人中的一人。治疗，如羟基脲（HU），诱导 胎儿血红蛋白（HbF）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？珠蛋白基因对这些疾病是姑息性的，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具有负面的副作用， 长期使用会致癌。此外，它是唯一有效的治疗SCD的方法 在过去的十年里发展起来的。人类类珠蛋白基因转换的发育调节由以下因素控制： 几个参数，主要是反式作用的转录环境和顺式作用的DNA元件。解开 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，Y编码样蛋白（TSPYL 1）和胎儿珠蛋白诱导因子（FGIF或ANKRD 49）， 被证明是上调的珠蛋白基因表达本提案的总体目标是探讨 这两种蛋白质作为治疗靶点在治疗SCD中的有用性， 作用，因此最终可以开发针对这些蛋白质调节的新疗法。为 具体目标1和2，我们将使用强制表达（功能获得）或表达敲低（功能丧失） 功能），以确定体内表型或发育作用。我们 将采用染色质免疫沉淀（ChIP）来确定？珠蛋白基因， 含有TSPYL 1或FGIF的复合物结合，并鉴定TSPYL 1或FGIF相互作用的伴侣蛋白 使用免疫沉淀（IP）结合质谱法。不太可能所有种类的 蛋白质参与？珠蛋白基因激活已经被发现，留下了许多更有益的治疗方法， 仍有待发现的目标。因此，对于具体目标3，我们将采用一种新的选择系统， 激活A？珠蛋白启动子-绿色荧光蛋白（GFP）融合在珠蛋白基因座酵母人工 来自转基因小鼠的染色体（<$-YAC）骨髓细胞（BMC），以鉴定新的反式激活因子 什么？珠蛋白合成，可能或可能不与TSPYL 1或FGIF合作。完成这些研究将 为进一步了解TSPYL 1和FGIF的功能提供了重要的动物模型和生化数据 在上调？珠蛋白基因在发育过程中的表达。该提案将产生独特的工具， 新的策略来了解？用于治疗SCD的珠蛋白基因调控。

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 ?-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. It is improbable that all classes of proteins involved in ?-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 ?-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.