Transactivation of Fetal Hemoglobin

胎儿血红蛋白的反式激活

• 批准号: 8010797
• 负责人: KENNETH R PETERSON
• 金额: $ 9.97万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-18 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010797
• 关键词: Adult; Adverse effects; Affect; African American; Animal Model; Ankyrin Repeat; Antibodies; Binding; Binding Sites; Biochemical; Bioinformatics; Bone Marrow Cells; Cells; Chemicals; Chromosomes, Artificial, Yeast; Complementary DNA; Complex; Conceptus; Coupled; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Data; Development; Dimerization; Disease; Elements; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Expression Library; Fetal Hemoglobin; Fetal Liver; Fluorescence-Activated Cell Sorting; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Globin; Goals; Green Fluorescent Proteins; Hemoglobinopathies; Hereditary Disease; Human; Immunoprecipitation; K562 Cells; Left; Link; Mass Spectrum Analysis; Measures; Mediating; Megakaryocytes; Messenger RNA; Molecular; Molecular Profiling; Molecular Weight; Mus; N-terminal; Nuclear Extract; Nucleosomes; Patients; Pattern; Phenotype; Protein Binding; Proteins; Recruitment Activity; Regulation; Reporter; Research; Role; Sickle Cell; Sickle Cell Anemia; Specificity; Staging; Structure; Switch Genes; System; Testing; Therapeutic; Therapeutic Intervention; Tissues; Trans-Activators; Transactivation; Transcriptional Activation; Transgenic Mice; Transgenic Organisms; Yang; base; blood vessel occlusion; cDNA Expression; chromatin immunoprecipitation; crosslink; effective therapy; fetal; fetal globin; gain of function; gel mobility shift assay; hematopoietic tissue; human TSPY protein; hydroxyurea; in vivo; knock-down; loss of function; meetings; mouse model; novel; palliative; prevent; promoter; protein complex; protein function; public health relevance; research study; therapeutic target; tool; transcription factor

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.

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；每年出生的400名非裔美国人中就有一人受到影响。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好处，因为持续表达？-珠蛋白基因可以缓解这些疾病，可能通过防止红细胞（RBC）镰状细胞和随后的血管闭塞。然而，胡芦苏有不良副作用，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治疗SCD的方法。人类的发育规律？类珠蛋白基因开关受几个参数控制，主要是反式作用的转录环境和顺式作用的DNA元件。揭示珠蛋白基因表达的潜在控制机制，特别是那些参与激活的？-珠蛋白合成对于识别治疗干预的新靶点很重要。人类蛋白，睾丸特异性蛋白，y编码样蛋白（TSPYL1）和胎儿球蛋白诱导因子（FGIF或ANKRD49），已被证明上调？-珠蛋白基因表达。本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SCD的治疗靶点的有用性，从而最终开发出针对这些蛋白调节的新疗法。对于Specific Aims 1和2，我们将在红系细胞中使用TSPYL1和FGIF的强制表达（功能获得）或敲低表达（功能丧失）来确定体内表型或发育影响。我们将采用染色质免疫沉淀（ChIP）来确定TSPYL1或FGIF复合物结合的3-球蛋白基因附近的序列，并使用免疫沉淀（IP）结合质谱法鉴定TSPYL1或FGIF与之相互作用的伴侣蛋白。这两种蛋白质的特异性是什么？-珠蛋白基因活化将被评估。参与3-珠蛋白基因激活的所有种类的蛋白质都已被发现是不可能的，还有许多有益的治疗靶点有待发现。因此，对于Specific Aim 3，我们将采用一种基于激活a ？-珠蛋白启动子-绿色荧光蛋白（GFP）在？-珠蛋白位点酵母人工染色体(？-YAC)骨髓细胞（BMCs），以鉴定可能与TSPYL1或FGIF结合或不结合的新的3-球蛋白合成反激活因子。这些研究的完成将为进一步了解TSPYL1和FGIF在上调？-珠蛋白基因在发育中的表达。这一建议将产生独特的工具和新的策略来理解？-珠蛋白基因调控治疗SCD。公共卫生相关性：镰状细胞病（SCD）是一种常见的遗传性疾病，影响着全世界数百万人。每年出生的500名非裔美国人中就有一人受到SCD的影响。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治疗方法，特别是针对打开胎儿3-珠蛋白基因的研究，这已被证明对治疗SCD有效。