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

项目总结： 胎儿到成人的血红蛋白在出生前后的转换涉及从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改善镰状细胞性贫血和β-地中海贫血。 因此，胎儿血红蛋白(Hbf)诱导物的鉴定是一个活跃的研究领域，具有巨大的研究价值。 治疗潜力。与转录和表观遗传调控不同，β的转录后调控- 人们对珠蛋白转换知之甚少，很少有关于其生理学和临床相关性的报道。我们的预赛 研究发现Pumilo-1(PUM1)是一种RNA结合蛋白，以前没有报道过在 红细胞生成，作为β-珠蛋白转换的转录后调节。PUM1，其表达受调控 红系主要转录因子EKLF/KLF1在红系高峰期表达 分化，结合胎儿γ-珠蛋白mRNA，降低γ-珠蛋白(HBG1mRNA)的稳定性和翻译 效率，最终导致γ-珠蛋白水平降低。PUM1被压低导致强劲增长 在胎儿γ-珠蛋白(约占总血红蛋白的22%)中，不影响成人红系细胞中的β-珠蛋白水平。 重要的是，敲除PUM1并不会限制红细胞生成的进展，提供了一种潜在的安全和 镰状细胞性贫血和β地中海贫血的有效治疗策略。为了支持这一想法，我们报告提升 在无贫血的情况下胎儿血红蛋白水平，在一个新的杂合子PUM1突变的个体中 RNA结合域(P.(His1090Pros*16)；c.3267_3270delTCAC)，表明PUM1介导的后 转录调控在人类血红蛋白转换过程中起着关键作用。基于这些初步的 结果：我们假设，损害成人红系细胞PUM1的RNA结合功能可以 诱导胎儿血红蛋白水平，对红细胞生成的其他方面没有有害影响；这种诱导可能 改善培养的镰状细胞贫血红系细胞的镰状等疾病表型。为了测试这一点 假设，我们提出了以下目标：1)破译使PUM1介导的调控元件 人红系细胞中胎儿血红蛋白沉默的研究2)PUM1基因敲除对人红系的影响 分化，3)评价PUM1基因敲除对镰状细胞性贫血红细胞性贫血的抑制作用 细胞。 我们的研究将首次描述PUM1在血红蛋白转换和转录后功能。 红细胞生成和揭示一个新的人类PUM1突变p.(His1090Pros*16)的影响 在一名HbF升高的患者身上发现。此外，这些研究将促进我们对 人类胎儿血红蛋白转录后沉默，人们对此知之甚少。最后，由于PUM1 作为细胞质转录后调节因子，我们的研究将确定是否干扰PUM1来诱导 胎儿血红蛋白可作为改善β的潜在非基因改变治疗靶点。 地中海贫血和镰状细胞性贫血。

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.