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

描述(由申请人提供)：了解人类γ-toβ-珠蛋白基因开关的分子机制在治疗诸如镰状细胞病、库利氏贫血和β地中海贫血等血红蛋白疾病方面一直被认为是重要的，因为大量证据表明，增加的胎儿血红蛋白(Hbf)显著降低了患者与这些疾病相关的病理生理。我们在这项研究中的目标是了解转录因子的翻译后修饰(PTM)在成人最终红细胞生成过程中参与γ-珠蛋白基因沉默的作用。我们的临床目标是根据这项研究的结果确定新的分子靶点，这些靶点可以通过治疗来调节γ-珠蛋白的合成，从而治疗这些疾病。我们最近证明，γ-珠蛋白沉默的一种模式发生在GATA结合位点上，分别位于Aγ-珠蛋白或Gγ-珠蛋白基因CAP位点的-566或-567位，并且 通过DNA结合部分GATA-1及其招募的共抑制物伙伴FOG-1和Mi2，NuRD染色质重塑复合体的一个组成部分。转录因子的翻译后修饰可能在调节转录抑制复合体或激活复合体的形成中发挥关键作用，特别是当这两种类型的复合体可能利用相同的DNA结合蛋白时，就像红系细胞中的GATA-1一样。我们认为，O-GlcN酰化(O-GlcNAc)PTM通过控制γ-珠蛋白基因上游GATA-1-FOG-1-MI2(NuRD)转录抑制物复合体的组装，参与调控γ-珠蛋白的转录。一个特定的目的将检验这样的假设，即GATA-1-FOG-1-Mi2(NuRD)转录/染色质重塑因子的O-GlcN酰化调节它们参与多蛋白抑制物复合体的参与。在特定的目标1a中，我们将研究O-GlcN酰化对γ-珠蛋白基因的调节。染色质免疫沉淀(ChIP)分析将被用来检测O-GlcNAc循环酶、O-GlcNAc转移酶(OGT)和O-GlcNAc酶(OgA)的时空排列、Aγ-珠蛋白启动子上的GATA-1-FOG-1-Mi2轴，以及在有或没有OGT/OGA基因敲除或OGA抑制的情况下，或在β-YAC转基因小鼠中，在终末分化或诱导珠蛋白基因表达的细胞培养中，或在有或没有OGT/OGA基因敲除或OGA抑制的情况下，细胞培养中O-GlcNAc的状态。在特定的目标1b中，我们将确定O-GlcNAc是否调节β样珠蛋白基因在红系特异性OGT条件性敲除β-YAC小鼠体内红系发育过程中的表达，或者在OGT或OGA强制表达的β-YAC双转基因(双基因)小鼠中。我们提出的研究代表了珠蛋白基因转换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，针对这些靶点，可以开发高度特异性的治疗方法，以增加HbF用于治疗这些红细胞疾病，而不会出现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.