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）：镰状细胞疾病（SCD）是一种常见的遗传疾病，影响了全球数百万的人；它影响每年出生的400名非裔美国人之一。诱导胎儿血红蛋白（HBF）的羟基脲（HU）等疗法对患有这种血红蛋白疾病的患者具有巨大的好处，因为持续表达了？ - 格珠蛋白基因对这些疾病的持续表达是对这些疾病的粘性，这可能是通过防止红细胞（RBC）（RBC）患病和随后的失血来吸收的。但是，HU具有负面影响，并且长期使用可能具有致癌性。此外，它独自一人是过去十年中SCD的唯一有效治疗方法。人类的发育调节？类似球蛋白基因转换由多个参数控制，主要是反式转录环境和顺式作用DNA元素。揭示球蛋白基因表达的控制机制，尤其是参与？ - 球蛋白合成的机制对于辨别治疗干预的新靶标很重要。人蛋白，睾丸特异性蛋白，Y编码样（TSPYL1）和胎儿球蛋白诱导因子（FGIF或ANKRD49）已显示出上调？ - 斑蛋白基因表达。该提案的总体目标是探索这两种蛋白质作为治疗靶标在治疗SCD方面的有用性，以确定其作用机制，以便最终可以开发出针对调节这些蛋白质的新疗法。对于特定目的1和2，我们将使用tspyl1和fGIF在红细胞中Tspyl1和FGIF的表达（功能丧失）的强制表达（功能丧失）来确定体内的表型或发育效应。我们将采用染色质免疫沉淀（CHIP）来确定含Tspyl1-或FGIF的复合物结合的3-珠蛋白基因附近的序列，并鉴定TSPYL1或FGIF使用免疫沉淀（IP）与质谱率相关的伴侣蛋白。 [将评估这两种蛋白质的特异性 - 珠蛋白基因的激活。]不可能发现所有参与3-氯丁蛋白基因激活的蛋白质的特异性，这是不可能发现的，这会发现仍有更多有益的治疗靶标。 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.这些研究的完成将提供重要的动物模型和生化数据，以进一步了解tspyl1和fGIF在发育过程中上调的？ - 斑蛋白基因表达中的功能。该提案将产生独特的工具和新策略，以了解用于治疗SCD的globin基因调节机制。公共卫生相关性：镰状细胞疾病（SCD）是一种常见的遗传疾病，影响了全球数百万的人。 SCD影响每年出生的500名非裔美国人之一。了解控制球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，特别是旨在打开胎儿3-蛋白基因的研究，这已被证明对SCD的治疗有效。