Post-Transcriptional Regulatory Mechanisms of Fetal Hemoglobin Repression

摘要：β-Hb病是最常见的遗传性单基因疾病之一，目前尚无治疗方法。 选择仍然有限。这些障碍的定义是无法培养出足够多的有功能的成年人 并有多种严重的健康后果。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，这种基因通常会随着时间的推移而被抑制 出生后的头几个月。然而，由于缺乏机械知识，治疗方法严重受阻。 关于胎儿血红蛋白抑制和血红蛋白转换。在这项工作中，我提议进行实验，以 研究成人和胎儿β类珠蛋白转录本的转录后调控。转录后 对γ-珠蛋白RNA的调控已经在多个水平上观察到。首先，Corfu基因缺失的生物学意义在于 显示了来自Corfu等位基因的γ-珠蛋白转录本的转录后沉默，尽管事实是 γ-珠蛋白的新转录是从所有Corfu缺失等位基因中上调的。此外，还观察到， 生理盐水处理CD34+细胞增强γ-珠蛋白的翻译而不影响转录本 HbB或HbG转录本的稳定性或定位。此外，对缺乏核的网织红细胞的研究有 尽管丁酸具有组蛋白抑制剂的已知作用，但它仍具有类似的作用 脱乙酰酶。最后，在最近的CRISPR筛查中，发现了20/117个HIT基因与HBF抑制物有关 核糖核酸结合、加工或腐烂。在这里，我提出了跟进DDX6的实验，即RNA结合 蛋白质最近在我们的实验室被确认为最有效的新的HBF抑制因子。而DDX6是 RNA加工和衰退的几个不同阶段，包括它作为P-体成分和作为 在miRNA介导的沉默中，DDX6在抑制胎儿血红蛋白中的作用仍然很大程度上尚不清楚。至 确定DDX6可能影响胎儿血红蛋白表达的机制，我将进行DDX6 ECLIP寻找可能与胎儿血红蛋白抑制相关的DDX6的RNA靶点。考虑到 DDX6作为P-体组分，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的DDX6相互作用蛋白的能力 经Hbf抑制物筛选鉴定，在存在或不存在DDX6的情况下直接与γ-珠蛋白转录本结合。 最后，我将使用反义寡核苷酸方法提取天然血红蛋白转录本，并分析RNA- 结合蛋白附着在每个转录本上。深入了解胎儿基因转录后调控 对血红蛋白和DDX6在胎儿血红蛋白抑制中的作用有可能确定新的靶点 β-地中海贫血、镰刀等β-Hb病的治疗干预 细胞病。

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.