Post-Transcriptional Regulatory Mechanisms of Fetal Hemoglobin Repression

胎儿血红蛋白抑制的转录后调节机制

• 批准号: 10019321
• 负责人: Steven Coyne
• 金额: $ 3.37万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019321
• 关键词: Adult; Affect; Alleles; Antisense Oligonucleotides; Binding; Binding Proteins; Biochemical; Biology; Birth; Butyrates; CD34 gene; CRISPR screen; Candidate Disease Gene; Cell Nucleus; Cells; Cellular biology; Characteristics; Cytoplasmic Granules; DNA Binding; Data; Development; Developmental Gene; Diagnosis; Disease; Erythroid; Erythroid Cells; Exhibits; Fetal Hemoglobin; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Globin; Health; Hemoglobin; Hemoglobin F Disease; Hemoglobinopathies; Heterozygote; Histone Deacetylase Inhibitor; Individual; Inherited; Investigation; Knock-out; Knowledge; Laboratories; Measures; Mediating; Mendelian disorder; Messenger RNA; Methods; MicroRNAs; Modeling; Outcome; Pathway interactions; Patients; Post-Transcriptional Regulation; Proteins; RNA; RNA Binding; RNA Decay; RNA Helicase; RNA Processing; RNA Stability; RNA metabolism; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Repression; Resolution; Reticulocytes; Role; Sickle Cell Anemia; Testing; Thalassemia; Therapeutic; Therapeutic Intervention; Time; Transcript; Translations; Work; beta Globin; beta Thalassemia; experimental study; fetal; follow-up; gamma Globin; insight; new therapeutic target; novel; novel therapeutic intervention; transcription factor; transcriptome

Abstract: β-Hemoglobinopathies are among the most common inherited monogenic disorders, yet treatment options remain limited. These disorders are defined by an inability to produce enough functional adult hemoglobin and have a variety of severe health outcomes. One major therapeutic approach which has proven successful is to raise the expression level of fetal hemoglobin, a gene typically repressed over time during the first months after birth. However, treatment methods are severely impeded by a lack of mechanistic knowledge regarding fetal hemoglobin repression and hemoglobin switching. In this work, I propose experiments to investigate the post-transcriptional regulation of adult and fetal β-type globin transcripts. Post-transcriptional regulation of γ-globin RNA has been observed at multiple levels. First, the biology of the Corfu deletion has shown post-transcriptional silencing of γ-globin transcripts derived from the Corfu allele despite the fact that new transcription of γ-globin is elevated from all Corfu deletion alleles. Additionally, it has been observed that treatment of CD34+ cells with salubrinal enhances the translation of γ-globin without affecting the transcript stability or localization of either HBB or HBG transcripts. Further, studies in reticulocytes which lack nuclei have demonstrated the capacity of butyrate to have similar effects despite its known role as an inhibitor of histone deacetylases. Finally, in a recent CRISPR screen to identify HbF repressors, 20/117 hit genes were associated with RNA binding, processing, or decay. Here, I propose experiments to follow up on DDX6, the RNA-binding protein recently identified in our laboratory as the most potent novel HbF repressor. While DDX6 is central to several distinct stages of RNA processing and decay including its role as a P-body constituent and as part of miRNA-mediated silencing, the role of DDX6 in repression of fetal hemoglobin remains largely unexplored. To identify mechanisms through which DDX6 may influence fetal hemoglobin expression, I will perform DDX6 eCLIP to identify RNA targets of DDX6 which may be relevant to fetal hemoglobin repression. Given the role of DDX6 as a P-body constitutent, I will assess the subcellular localization of DDX6 protein and globin transcripts in adult and fetal erythroid models. Further, I aim to directly test the ability of known DDX6 interacting proteins identified by HbF repressor screen to bind directly to γ-globin transcripts in the presence or absence of DDX6. Finally, I will use anti-sense oligo methods to pull down native hemoglobin transcripts and profile the RNA- binding proteins attached to each transcript. Gaining insight into the post-transcriptional regulation of fetal hemoglobin and to the role of DDX6 in fetal hemoglobin repression has the potential to identify new targets for therapeutic intervention for patients diagnosed with β-Hemoglobinopathies including β-Thalassemia and Sickle Cell Disease.

摘要：β-血红蛋白病是最常见的遗传性单基因疾病之一， 选择仍然有限。这些疾病的定义是无法产生足够的功能性成人 血红蛋白，并有各种严重的健康后果。一种主要的治疗方法已经证明 成功的方法是提高胎儿血红蛋白的表达水平，这是一种在胎儿发育过程中随时间推移而被抑制的基因。 出生后的第一个月。然而，由于缺乏机械知识，治疗方法受到严重阻碍 关于胎儿血红蛋白抑制和血红蛋白转换。在这项工作中，我提出的实验， 研究成人和胎儿β型珠蛋白转录本的转录后调控。转录后 已经在多个水平上观察到γ-珠蛋白RNA的调节。首先，科孚岛缺失的生物学特征 显示来自Corfu等位基因的γ-珠蛋白转录物的转录后沉默，尽管事实上 γ-珠蛋白的新转录从所有Corfu缺失等位基因升高。此外，据观察， 用salubrinal处理CD 34+细胞增强γ-珠蛋白的翻译而不影响转录 HBB或HBG转录物的稳定性或定位。此外，对缺乏细胞核的网织红细胞的研究表明， 证明了丁酸盐具有类似作用的能力，尽管其已知的作用是组蛋白抑制剂 脱乙酰酶最后，在最近的一项识别HbF阻遏物的CRISPR筛选中，117个命中基因中有20个与HbF阻遏物相关。 RNA的结合、加工或衰变。在这里，我提出了对DDX6的后续实验， 最近在我们的实验室中鉴定为最有效的新型HbF阻遏物的蛋白。虽然DDX6是核心， RNA加工和衰变的几个不同阶段，包括其作为P体成分的作用和作为 在miRNA介导的沉默中，DDX6在抑制胎儿血红蛋白中的作用仍然在很大程度上未被探索。到 为了确定DDX6可能影响胎儿血红蛋白表达的机制，我将进行DDX6 eCLIP以鉴定可能与胎儿血红蛋白抑制相关的DDX6的RNA靶点。鉴于 DDX6作为P体的一个组成部分，我将评估DDX6蛋白和珠蛋白转录本的亚细胞定位 在成人和胎儿红系模型中。此外，我的目标是直接测试已知DDX6相互作用蛋白的能力， 通过HbF阻遏物筛选鉴定，在存在或不存在DDX6的情况下直接结合γ-珠蛋白转录物。 最后，我将使用反义寡核苷酸方法来拉下天然血红蛋白转录本，并分析RNA- 与每个转录物相连的结合蛋白。深入了解胚胎发育的转录后调控 DDX6在胎儿血红蛋白抑制中的作用有可能确定新的靶点， 对诊断为β-血红蛋白病（包括β-地中海贫血和镰状贫血）患者的治疗干预 细胞疾病。