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，但治疗 选项仍然有限。这些疾病的定义是无法产生足够的成年人 血红蛋白具有多种严重的健康结果。证明的一种主要治疗方法 成功的是提高胎儿血红蛋白的表达水平，该基因通常会随着时间的流逝而反映 出生后的头几个月。但是，缺乏机械知识，严重阻碍了治疗方法 考虑胎儿血红蛋白反射和血红蛋白切换。在这项工作中，我建议 研究成人和胎儿β型球蛋白转录本的转录后调节。转录后 已经在多个水平上观察到γ-球蛋白RNA的调节。首先，Corfu缺失的生物学具有 显示了从Corfu等位基因目的地得出的γ-球蛋白转录本的转录后沉默。 从所有CORFU缺失等位基因中升高γ-球蛋白的新转录。另外，已经观察到 用salubrinal增强CD34+细胞的处理不影响转录本而不影响转录本 HBB或HBG转录本的稳定性或定位。此外，对缺乏核的网状细胞的研究 证明丁酸具有类似作用的能力降低了其已知的组蛋白抑制剂的作用 脱乙酰基酶。最后，在最近的CRISPR屏幕中识别HBF复制品，相关的20/117 HIT基因 带有RNA结合，加工或衰减。在这里，我建议在RNA结合的DDX6上进行跟进 蛋白质最近在我们的实验室中确定为最有效的新型HBF复制剂。而ddx6是 RNA处理和衰减的几个不同阶段，包括其作为P体的作用构成的作用，并作为 miRNA介导的沉默，DDX6在胎儿血红蛋白表达中的作用在很大程度上仍然是出乎意料的。到 确定DDX6可能影响胎儿血红蛋白表达的机制，我将执行DDX6 ECLIP鉴定可能与胎儿血红蛋白表达有关的DDX6的RNA靶标。鉴于 DDX6作为P体构成，我将评估DDX6蛋白和球蛋白转录本的亚细胞定位 在成人和胎儿红细胞模型中。此外，我的目标是直接测试已知DDX6相互作用蛋白的能力 在存在或不存在DDX6的情况下，由HBF复制器屏幕鉴定，直接与γ-球蛋白转录本结合。 最后，我将使用反义的寡寡核方法拉下天然血红蛋白转录本并介绍RNA- 连接到每个转录本上的结合蛋白。洞悉胎儿的转录后调节 血红蛋白和DDX6在胎儿血红蛋白表达中的作用具有鉴定新目标的潜力 针对诊断患有β-血红蛋白病的患者的治疗干预措施，包括β-丘脑和镰刀 细胞疾病。