Post-Transcriptional Regulatory Mechanisms of Fetal Hemoglobin Repression

胎儿血红蛋白抑制的转录后调节机制

基本信息

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
项目类别：
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

项目摘要

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，但治疗方法这些疾病的选择仍然有限。一种已被证明的主要治疗方法。成功的方法是提高胎儿血红蛋白的表达水平，该基因通常在怀孕期间随着时间的推移而受到抑制然而，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，我建议进行实验。研究成人和胎儿 β 型珠蛋白转录后的转录后调节。 γ-珠蛋白 RNA 的调节已在多个层面上被观察到。显示源自 Corfu 等位基因的 γ-珠蛋白转录物的转录后沉默，尽管事实上所有 Corfu 缺失等位基因的 γ-球蛋白新转录均升高。此外，还观察到：用 salubrinal 处理 CD34+ 细胞可增强 γ-珠蛋白的翻译而不影响转录本此外，对缺乏细胞核的网织红细胞进行了研究。尽管丁酸盐具有组蛋白抑制剂的已知作用，但其具有类似作用的能力最后，在最近鉴定 HbF 阻遏物的 CRISPR 筛选中，20/117 个命中基因被关联起来。在这里，我建议对 DDX6（RNA 结合）进行后续实验。我们实验室最近发现 DDX6 蛋白是最有效的新型 HbF 抑制蛋白。 RNA 加工和衰变的几个不同阶段，包括其作为 P 体成分和作为 miRNA 介导的沉默，DDX6 在抑制胎儿血红蛋白中的作用在很大程度上仍未被探索。确定 DDX6 可能影响胎儿血红蛋白表达的机制，我将执行 DDX6 鉴于 DDX6 的作用，eCLIP 可以识别可能与胎儿血红蛋白抑制相关的 RNA 靶点。 DDX6 作为 P 体构建体，我将评估 DDX6 蛋白和球蛋白转录本的亚细胞定位此外，我的目标是直接测试已知 DDX6 相互作用蛋白的能力。通过 HbF 阻遏物筛选鉴定出在存在或不存在 DDX6 的情况下直接与 γ-珠蛋白转录物结合。最后，我将使用反义寡核苷酸方法来拉取天然血红蛋白转录物并分析 RNA- 附着在每个转录本上的结合蛋白，深入了解胎儿的转录后调节。血红蛋白以及 DDX6 在胎儿血红蛋白抑制中的作用有可能确定新的靶标对诊断为 β 血红蛋白病（包括 β 地中海贫血和镰状细胞病）的患者进行治疗干预细胞疾病。