Investigating PUM1 mediated post-transcriptional regulation of human hemoglobin switching and erythropoiesis

研究 PUM1 介导的人血红蛋白转换和红细胞生成的转录后调节

• 批准号: 10568059
• 负责人: Merlin Nithya Gnanapragasam
• 金额: $ 42.58万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568059
• 关键词: 3' Untranslated Regions; Adult; Affect; Anemia; Area; Binding; Binding Sites; Birth; CRISPR/Cas technology; Cell Cycle; Cells; Chromatin; Clinical; Collaborations; Cytoplasm; Data; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Fetal Hemoglobin; Flow Cytometry; Gene Silencing; Genes; Genome; Globin; Hemoglobin; Hemoglobin concentration result; Hereditary Disease; Heterozygote; Human; Impairment; Individual; Mediating; Messenger RNA; Molecular; Mutation; New York; Nuclear; Oxygen; Patients; Physiological; Post-Transcriptional Regulation; Process; Proliferating; Proteins; Protocols documentation; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Regulatory Element; Reporting; Research; Role; Sickle Cell Anemia; Sickle Cell Trait; Site; Symptoms; Testing; Therapeutic; Time; Transcriptional Regulation; alpha Globin; beta Globin; beta Thalassemia; clinically relevant; disease phenotype; effective therapy; epigenetic regulation; erythroid Kruppel-like factor; erythroid differentiation; fetal; gamma Globin; genome editing; genome-wide analysis; knock-down; lentivirally transduced; mRNA Stability; mRNA Translation; novel; posttranscriptional; progenitor; programs; promoter; sickling; therapeutic target; transcription factor; transcriptome sequencing; treatment strategy

Project Summary: The fetal to adult hemoglobin switching around birth involves an expression shift from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Hence, identification of inducers of fetal hemoglobin (HbF) is an active area of research and holds immense therapeutic potential. Unlike the transcriptional and epigenetic regulation, post-transcriptional regulation of β- globin switching is poorly understood, with few reports on its physiological and clinical relevance. Our preliminary studies have identified Pumilo-1 (PUM1), an RNA binding protein with no previously reported functions in erythropoiesis, as a post-transcriptional regulator of β-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor, Erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds fetal γ-globin mRNA, and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal γ-globin (~22% HbF of total hemoglobin), without affecting adult β-globin levels in human erythroid cells. Importantly, knocking down PUM1 does not limit erythropoiesis progression, providing a potentially safe and effective treatment strategy in sickle cell anemia and β-thalassemia. In support of this idea, we report elevated fetal hemoglobin levels in the absence of anemia, in an individual with a novel heterozygous PUM1 mutation in the RNA binding domain (p.(His1090Profs*16); c.3267_3270delTCAC), suggesting that PUM1 mediated post- transcriptional regulation is a critical player during human hemoglobin switching. Based on these preliminary results, we hypothesize that impairing the RNA binding functions of PUM1 in human adult erythroid cells can induce fetal hemoglobin levels, without deleterious effects on other aspects of erythropoiesis; this induction could ameliorate the disease phenotypes such as sickling in cultured sickle cell anemia erythroid cells. To test this hypothesis, we propose the following aims: 1) Decipher the regulatory elements that enable PUM1 mediated fetal hemoglobin silencing in human erythroid cells, 2) Study the impact of PUM1 knockdown on human erythroid differentiation, 3) Evaluate the capacity of PUM1 knockdown to relieve sickling in sickle cell anemia erythroid cells. Our studies will for the first time describe the post-transcriptional functions of PUM1 in hemoglobin switching and erythropoiesis and reveal the impact of a novel human PUM1 mutation p.(His1090Profs*16), which we have identified in a patient with elevated HbF. Further, these studies will advance our mechanistic understanding of the post-transcriptional silencing of human fetal hemoglobin, which is poorly understood. Finally, since PUM1 functions as a cytoplasmic post-transcriptional regulator, our studies will determine if disrupting PUM1 to induce fetal hemoglobin could serve as a potential non-gene altering therapeutic target towards ameliorating β- thalassemia and sickle cell anemia.

项目摘要： 胎儿到成人血红蛋白在出生前后的转换涉及到γ-珠蛋白到β-珠蛋白的表达转变， 红系细胞胎儿γ-珠蛋白的有效再表达可以改善镰状细胞贫血和β-地中海贫血。 因此，胎儿血红蛋白（HbF）诱导剂的鉴定是一个活跃的研究领域， 治疗潜力与转录和表观遗传调控不同，β- 球蛋白转换的了解很少，很少有关于其生理和临床相关性的报道。我们的初步 研究已经鉴定了Pumilo-1（Pumilo-1），一种RNA结合蛋白，其先前没有报道过的功能， 红细胞生成，作为β-珠蛋白转换的转录后调节因子。1，其表达受调控 红系主要转录因子，红系Krüppel样因子（EKLF/KLF 1），在红系 分化，结合胎儿γ-球蛋白mRNA，并降低γ-球蛋白（HBG 1）mRNA的稳定性和翻译 效率，其在降低的γ-珠蛋白蛋白水平中达到顶点。敲除B11导致了一个强劲的增长 在胎儿γ-珠蛋白（约占总血红蛋白的22% HbF）中，不影响人红系细胞中成人β-珠蛋白水平。 重要的是，敲低RIP 1并不限制红细胞生成进展，提供了一种潜在的安全和有效的治疗方法。 镰状细胞性贫血和β-地中海贫血有效治疗策略。为了支持这一观点，我们报告了 在没有贫血的情况下，胎儿血红蛋白水平，在一个新的杂合突变的个体， RNA结合域（p.（His 1090 Profs *16）; c.3267_3270delTCAC），表明His 11介导的后- 转录调节在人血红蛋白转换过程中是关键的参与者。基于这些初步 结果，我们假设，在人成人红系细胞中， 诱导胎儿血红蛋白水平，而对红细胞生成的其他方面没有有害影响;这种诱导可以 改善疾病表型，如培养的镰状细胞性贫血红系细胞中的镰状化。为了验证这一 假设，我们提出了以下目标：1）破译的调控元件，使EST 1介导的 人红系细胞中的胎儿血红蛋白沉默，2）研究RMB 1敲低对人红系细胞的影响 3）评估RMB 1敲低减轻镰状细胞性贫血红系中镰状化的能力 细胞 我们的研究将第一次描述在血红蛋白转换中， 红细胞生成，并揭示了一种新的人类p111突变的影响。（His 1090 Profs *16），我们有 在HbF升高的患者中鉴定。此外，这些研究将促进我们对 人类胎儿血红蛋白的转录后沉默，这是知之甚少。最后，自2001年以来， 作为一个细胞质转录后调节因子，我们的研究将确定是否破坏BMP 1诱导 胎儿血红蛋白可以作为一个潜在的非基因改变治疗靶点，以改善β- 地中海贫血和镰状细胞性贫血。