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

描述（由申请人提供）：长期以来，人们一直认为理解人类γ-至β-珠蛋白基因开关的分子机制对于血红蛋白病（如镰状细胞病（SCD）、库利贫血和β-地中海贫血）的治疗非常重要，因为大量证据表明，胎儿血红蛋白（HbF）增加显著降低了患者中与这些疾病相关的病理生理学。本研究的目的是了解γ-珠蛋白基因沉默过程中转录因子翻译后修饰（PTMs）的作用。我们的临床目标是基于本研究的结果来鉴定新的分子靶点，其可以在治疗上被调节以上调γ-珠蛋白合成来治疗这些疾病。我们最近证明，γ-珠蛋白沉默的一种模式发生在相对于Aγ-珠蛋白或Gγ-珠蛋白基因CAP位点分别位于-566或-567的加塔结合位点，并且是 通过DNA结合部分加塔-1及其共阻遏物伙伴FOG-1和Mi2（NuRD染色质重塑复合物的组分）的募集介导。转录因子的翻译后修饰可能在调节转录阻遏物复合物或激活物复合物的形成中起关键作用，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，如红系细胞中的加塔-1的情况。我们认为O-GlcNAc PTM通过控制γ-珠蛋白基因上游加塔-1-FOG-1-Mi2（NuRD）转录抑制复合物的组装参与调节γ-珠蛋白的转录。单个特定目的将检验以下假设：加塔-1-FOG-1-Mi2（NuRD）转录/染色质重塑因子的O-GlcNAc化调节其参与多蛋白阻遏物复合物。在特定目标1a中，我们将研究O-GlcNAc化对γ-珠蛋白基因的调控。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-GlcNAc酶（OGA）的空间和时间排列、Aγ-珠蛋白启动子处蛋白质的加塔-1-FOG-1-Mi2轴以及终末分化或诱导珠蛋白基因表达后细胞培养物中启动子本身的O-GlcNAc状态，在有或没有OGT/OGA敲低或OGA抑制的情况下，或在胎肝定形红细胞生成期间的β-YAC转基因小鼠中。在特定目的1b中，我们将确定在红系特异性floxed OGT条件性敲除β-YAC小鼠或OGT或OGA强制表达β-YAC双转基因（双基因）小鼠体内红细胞生成发育期间，O-GlcNAc是否调节β-样珠蛋白基因表达。我们提出的研究代表了珠蛋白基因开关领域的一条新的研究途径。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，用于治疗这些红细胞疾病，而不会产生与广谱治疗相关的副作用。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms underlying the human γ- to β-globin gene switch has long been recognized as important in the treatment of hemoglobinopathies such as sickle cell disease (SCD), Cooley's anemia and β-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 γ-globin gene silencing during the adult definitive erythropoiesis. Our clinical goal is to identify new molecular targets based on the outcome of this study that can be modulated therapeutically for up-regulation of γ-globin synthesis to treat these diseases. We recently demonstrated that one mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-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 γ-globin transcription by controlling assembly of the GATA-1-FOG-1-Mi2 (NuRD) transcriptional repressor complex upstream of the γ-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 γ-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 Aγ- 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 β-YAC transgenic mice during fetal liver definitive erythropoiesis. In Specific Aim 1b, we will determine whether O-GlcNAc regulates β-like globin gene expression during erythropoietic development in vivo in erythroid-specific floxed OGT conditional knockout β-YAC mice, or in OGT or OGA enforced-expression β-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.