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）水平升高可显著改善β-半乳糖不耐症患者的临床结局。 血红蛋白病，如镰状细胞病（SCD）。FDA唯一批准的治疗SCD的药物是 羟基脲，其通过上调HbF起作用。然而，其疗效在不同患者中是可变的 其作用机制尚不清楚。因此，确定上调HbF的方式，如 抑制HbF阻遏物是该领域的长期兴趣。BCL 11 A和LRF是转录因子， 与相关的辅助调节因子一起独立地发挥作用以抑制HbF，但在治疗潜力方面具有局限性。 虽然这些转录因子和它们的共调节因子已经被广泛研究，但转录因子的上游调节仍然是一个重要的研究课题。 这些转录因子，如转录后调节，没有得到很好的研究。阐明这些 未知的机制可能会发现新的治疗靶点，可以绕过这些限制， 主要HbF阻遏物保持不变。为此，我采用了一种基于CRISPR/Cas9的筛选方法， RNA结合蛋白（RBP）在HbF基因调控中的作用。 使用HUDEP 2细胞，一种人类红系祖细胞系，我们询问了527个人类RBP，发现 几种限制性商业惯例被称为异质核限制性商业惯例， 核糖核蛋白（hnRNP）显着上调HbF。在这些蛋白质中，具有最高效果的候选物 大小为synaptotagmin结合胞质RNA相互作用蛋白（SYNCRIP）。我们验证了这一结果， 使用CRISPR/Cas9敲低HUDEP 2细胞中的SYNCRIP并通过流式细胞术评估HbF水平 流式细胞术、蛋白质印迹和RT-qPCR。我们发现，在SYNCRIP敲低后，HbF表达被抑制。 在转录和蛋白质水平上不影响BCL 11 A或LRF。 hnRNP是一类对转录后调控的多个方面都很重要的RBP， 例如前mRNA剪接、mRNA转运、稳定化和翻译。目前，hnRNP尚未被 涉及HbF抑制，并且在造血背景下对SYNCRIP的研究有限。我的目标是 阐明SYNCRIP和其他类型的hnRNP调节HbF基因表达的机制。 我假设SYNCRIP与其他hnRNP沿着调节转录物的RNA加工 编码与BCL 11 A或LRF相关的共调节子。在目标1中，我将研究SYNCRIP的RNA在 结合活性在调节HbF表达中起作用。值得注意的是，已知两个额外的hnRNP相互调节 也在这个屏幕上被识别出来。因此，在目标2中，我将研究这些hnRNP的合作机制 HbF调节。 通过成功地完成这些目标，我将获得关于这种新型HbF模型的进一步信息 抑制，其可以潜在地用于缓解SCD的治疗目的。