Elucidating mechanisms of hnRNPs in fetal hemoglobin regulation

阐明 hnRNP 在胎儿血红蛋白调节中的机制

• 批准号: 10192713
• 负责人: AOI WAKABAYASHI
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10192713
• 关键词: Affect; Alternative Splicing; Binding; Blood Vessels; Bypass; CRISPR screen; CRISPR/Cas technology; Categories; Cell Line; Cells; Clinical; Complementary DNA; Data; Disease; Drug Targeting; Erythrocytes; Erythroid Cells; Erythroid Progenitor Cells; Event; FDA approved; Fetal Hemoglobin; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Globin; Goals; Health Benefit; Hematological Disease; Hematopoiesis; Hemoglobin; Hemoglobinopathies; Heterogeneous-Nuclear Ribonucleoproteins; Human; Inherited; Knock-out; Measures; Mediating; Messenger RNA; Modeling; NuRD complex; Outcome; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Play; Point Mutation; Polymers; Post-Transcriptional Regulation; Process; Protein Isoforms; Proteins; RNA; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Regulation; Repression; Reverse Transcriptase Polymerase Chain Reaction; Role; Sickle Cell; Sickle Cell Anemia; Site; Symptoms; Testing; Therapeutic; Transcript; Transcriptional Regulation; Translations; Validation; Western Blotting; Work; base; beta Globin; clinically significant; cofactor; cooperative study; experimental study; gamma Globin; hydroxyurea; improved; inhibitor/antagonist; interest; knock-down; mRNA Precursor; new therapeutic target; novel; overexpression; pain symptom; protein function; screening; synaptotagmin; therapeutic development; therapeutic target; transcription factor

Project Summary Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta- hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD is hydroxyurea, which works through upregulating HbF. However, its efficacy is variable among different patients and the mechanism of action is not well understood. Therefore, identifying ways of upregulating HbF, such as inhibiting HbF repressors, is a long-standing interest in this field. BCL11A and LRF are transcription factors that independently function with associated co-regulators to repress HbF, but have limitations in therapeutic potential. While these transcription factors and their co-regulators have been extensively studied, upstream regulation of these transcription factors, such as post-transcriptional regulation, are not well studied. Elucidating these unknown mechanisms may uncover novel therapeutic targets that can bypass the limitations targeting these major HbF repressors hold. To this end, I employed a CRISPR/Cas9 based screening approach to interrogate RNA binding proteins (RBP) in HbF gene regulation. Using HUDEP2 cells, a human erythroid progenitor cell line, we interrogated 527 human RBPs and found that depletion of several RBPs that belong to a category of RBPs termed heterogeneous nuclear ribonucleoproteins (hnRNP) significantly upregulate HbF. Of these proteins, the candidate with the highest effect size was synaptotagmin-binding cytoplasmic RNA interacting protein (SYNCRIP). We validated this result by knocking down SYNCRIP in HUDEP2 cells using CRISPR/Cas9 and assessing the levels of HbF via flow cytometry, western blot, and RT-qPCR. We found that upon SYNCRIP knock down, HbF expression was significantly increased without impacting BCL11A or LRF on the transcriptional and protein level. hnRNP is a category of RBPs that are important for multiple aspects of post transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. Currently, hnRNPs have not been implicated in HbF repression and studies on SYNCRIP in the context of hematopoiesis is limited. I aim to elucidate the mechanisms by which SYNCRIP and other types of hnRNPs work to regulate HbF gene expression. I hypothesize that SYNCRIP, along with other hnRNPs, work to regulate the RNA processing of transcripts encoding co-regulators associated with BCL11A or LRF. In aim 1, I will investigate the role SYNCRIP’s RNA binding activity plays in regulating HbF expression. Notably, two additional hnRNPs known to regulate each other were also identified in this screen. Therefore, in aim 2, I will study the cooperative mechanism of these hnRNPs in HbF regulation. By successfully completing these aims, I will have gained further information on this novel model of HbF repression, which can potentially be exploited for therapeutic purposes in alleviating SCD.

项目摘要 胎儿血红蛋白（HBF）水平升高可显着改善β- 血红蛋白病，例如镰状细胞病（SCD）。治疗SCD的唯一的FDA批准药物是 羟基脲，通过上调HBF起作用。但是，其效率在不同患者中是可变的 而且作用机理尚未得到充分理解。因此，确定上调HBF的方法，例如 抑制HBF复制品是对这一领域的长期兴趣。 Bcl11a和LRF是转录因素 与相关的共同调节器独立发挥作用以抑制HBF，但在治疗潜力方面有局限性。 尽管这些转录因子及其共同调节因子已广泛研究，但上游调节 这些转录因子（例如转录后调节）并不是很好的研究。阐明这些 未知机制可能会发现可以绕开针对这些的局限性的新型治疗靶标 主要的HBF复制品保持。为此，我在基于CRISPR/CAS9的筛选方法上进行了询问 HBF基因调节中的RNA结合蛋白（RBP）。 使用HUDEP2细胞，人类红细胞祖细胞系，我们询问了527人RBP并发现 属于称为异质核的一类RBP的几种RBP的耗竭 核糖核蛋白（HNRNP）显着上调HBF。在这些蛋白质中，效果最高的候选者 大小为突触蛋白结合的细胞质RNA相互作用蛋白（Syncrip）。我们通过 使用CRISPR/CAS9在HUDEP2细胞中拆卸syncrip，并通过流量评估HBF的水平 细胞仪，蛋白质印迹和RT-QPCR。我们发现合成后，HBF表达为 显着增加而没有影响BCL11A或LRF对转录和蛋白质水平的影响。 HNRNP是RBP的类别，对于转录后调节的多个方面很重要， 例如前mRNA剪接，mRNA转运，稳定和翻译。目前，hnrnps还没有 在HBF表达中实施和在造血中有关综合的研究是有限的。我的目标 阐明合成和其他类型的HNRNP调节HBF基因表达的机制。 我假设该合成以及其他HNRNP努力调节转录本的RNA处理 与BCL11A或LRF相关的编码共同调节器。在AIM 1中，我将调查Syncrip的RNA角色 结合活性在调节HBF表达中发挥作用。值得注意的是，已知互相调节的另外两个HNRNP 也在此屏幕上确定。因此，在AIM 2中，我将研究这些HNRNP的合作机制 在HBF调节中。 通过成功完成这些目标，我将获得有关HBF新颖模型的进一步信息 镇压，可以在减轻SCD的治疗目的中探索的抑制作用。