Regulation of Globin Gene Switching by O-GlcNAc Post-Translational Modification

O-GlcNAc 翻译后修饰调控球蛋白基因转换

• 批准号: 8610686
• 负责人: KENNETH R PETERSON
• 金额: $ 26.27万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8610686
• 关键词: Address; Adult; Adverse effects; Affect; African American; Binding Sites; Biochemical; Biological; Cell Culture Techniques; Cell Cycle; Cell physiology; Cells; Chromatin Remodeling Factor; Complex; Cooley' s anemia; Cytoplasmic Protein; DNA Binding; DNA-Binding Proteins; Data; Development; Disease; Drosophila genus; Enzymes; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Fetal Hemoglobin; Fetal Liver; Functional disorder; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Globin; Glucosamine; Goals; Hemoglobinopathies; Hereditary Disease; Homeobox Genes; Human; Knock-out; Knockout Mice; Knowledge; Lead; Mediating; Modeling; Modification; Molecular; Molecular Target; Mus; NuRD complex; Nuclear Proteins; O-GlcNAc transferase; Outcome; Outcome Study; Pathway interactions; Patients; Physiological; Play; Polycomb; Post-Translational Protein Processing; Promoter Regions; Proteins; Regulation; Relative (related person); Repression; Repressor Proteins; Research; Role; Serine; Sickle Cell Anemia; Site; Switch Genes; System; Testing; Thalassemia; Therapeutic; Threonine; Transcription Coactivator; Transcription Repressor/Corepressor; Transgenic Mice; Transgenic Organisms; Translations; Up-Regulation; base; biological adaptation to stress; chromatin immunoprecipitation; drug development; effective therapy; fetal; gain of function; gene repression; human GATA1 protein; in vivo; loss of function; mouse model; new therapeutic target; novel; null mutation; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; promoter; public health relevance; transcription factor

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms underlying the human ?- to ¿-globin gene switch has long been recognized as important in the treatment of hemoglobinopathies such as sickle cell disease (SCD), Cooley's anemia and ¿-thalassemias, since a wealth of evidence demonstrates that increased fetal hemoglobin (HbF) significantly decreases the pathophysiology associated with these diseases in patients. Our goal in this study is to understand the role of post-translation modifications (PTMs) of transcription factors involved in ?-globin gene silencing during the adult definitive erythropoiesis. Our clinica goal is to identify new molecular targets based on the outcome of this study that can be modulated therapeutically for up-regulation of ?-globin synthesis to treat these diseases. We recently demonstrated that one mode of ?-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the A?-globin or G?-globin gene CAP sites, respectively, and is mediated through the DNA-binding moiety GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi2, a component of the NuRD chromatin remodeling complex. Post-translational modifications of transcription factors may play a critical role in regulating the formation of transcriptional repressor complexes or activator complexes, particularly when both types of complexes might utilize the same DNA-binding protein, as is the case for GATA-1 in erythroid cells. We propose that the O-GlcNAcylation (O-GlcNAc) PTM is involved in regulating ?-globin transcription by controlling assembly of the GATA-1-FOG-1-Mi2 (NuRD) transcriptional repressor complex upstream of the ?-globin genes. A single Specific Aim will test the hypothesis that O-GlcNAcylation of GATA-1-FOG-1-Mi2 (NuRD) transcription/chromatin remodeling factors regulates their participation in a multi-protein repressor complex. In Specific Aim 1a, we will investigate the regulation of the ?-globin genes by O-GlcNAcylation. Chromatin immunoprecipitation (ChIP) analyses will be utilized to examine the spatial and temporal arrangement of the O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), the GATA-1-FOG-1-Mi2 axis of proteins at the A?- globin promoter and the O-GlcNAc status of the promoter itself in cell cultures following terminal differentiation or induction of globin gene expression, with or without OGT/OGA knockdown or OGA inhibition, or in ¿-YAC transgenic mice during fetal liver definitive erythropoiesis. In Specific Aim 1b, we will determine whether O-GlcNAc regulates ¿-like globin gene expression during erythropoietic development in vivo in erythroid-specific floxed OGT conditional knockout ¿-YAC mice, or in OGT or OGA enforced-expression ¿-YAC bi-transgenic (bigenic) mice. Our proposed studies represent a new avenue of research in the globin gene switching field. The knowledge we gain from these studies will reveal novel therapeutic targets for which highly-specific treatments may be developed to increase HbF for the treatment of these red blood cells diseases without the side-effects associated with broad-spectrum therapies.

描述（由申请人提供）：了解人类 α- 至 β- 珠蛋白基因转换的分子机制长期以来被认为对于治疗血红蛋白病（如镰状细胞病 (SCD)、库利氏贫血和 β-地中海贫血）非常重要，因为大量证据表明胎儿血红蛋白 (HbF) 的增加可显着降低与这些疾病相关的病理生理学。 患者的疾病。我们本研究的目标是了解成人确定性红细胞生成过程中涉及 β-珠蛋白基因沉默的转录因子翻译后修饰 (PTM) 的作用。我们的临床目标是根据本研究的结果确定新的分子靶点，可以通过治疗性调节来上调 β-珠蛋白合成来治疗这些疾病。我们最近证明，一种模式的α-珠蛋白沉默发生在分别相对于Aβ-珠蛋白或Gβ-珠蛋白基因CAP位点位于-566或-567的GATA结合位点，并且是 通过 DNA 结合部分 GATA-1 及其共阻遏物伴侣 FOG-1 和 Mi2（NuRD 染色质重塑复合物的组成部分）的募集来介导。转录因子的翻译后修饰可能在调节转录阻遏复合物或激活复合物的形成中发挥关键作用，特别是当两种类型的复合物可能利用相同的 DNA 结合蛋白时，如红系细胞中的 GATA-1 的情况。我们提出 O-GlcNAc 修饰 (O-GlcNAc) PTM 通过控制 β-珠蛋白基因上游 GATA-1-FOG-1-Mi2 (NuRD) 转录抑制复合物的组装来参与调节 β-珠蛋白转录。单一特定目标将检验 GATA-1-FOG-1-Mi2 (NuRD) 转录/染色质重塑因子的 O-GlcNAc 酰化调节其参与多蛋白阻遏复合物的假设。在具体目标 1a 中，我们将研究 O-GlcNAc 酰化对 β-珠蛋白基因的调节。染色质免疫沉淀 (ChIP) 分析将用于检查 O-GlcNAc 循环酶、O-GlcNAc 转移酶 (OGT) 和 O-GlcNAcase (OGA) 的空间和时间排列、Aβ-珠蛋白启动子处蛋白质的 GATA-1-FOG-1-Mi2 轴以及细胞培养物中启动子本身的 O-GlcNAc 状态 在终末分化或诱导珠蛋白基因表达后，有或没有 OGT/OGA 敲低或 OGA 抑制，或在 ¿-YAC 转基因小鼠中在胎儿肝脏确定性红细胞生成过程中。在具体目标 1b 中，我们将确定在红细胞特异性 floxed OGT 条件敲除 ¿-YAC 小鼠或 OGT 或 OGA 强制表达 ¿-YAC 双转基因（双基因）小鼠体内红细胞生成发育过程中，O-GlcNAc 是否调节 ¿ 样珠蛋白基因表达。我们提出的研究代表了珠蛋白基因转换领域的新研究途径。我们从这些研究中获得的知识将揭示新的治疗靶点，可以开发高度特异性的治疗方法来增加 HbF 来治疗这些红细胞疾病，而不会产生与广谱疗法相关的副作用。