Elucidating mechanisms of hnRNPs in fetal hemoglobin regulation

项目摘要 胎儿血红蛋白(HBF)水平升高可显著改善贝塔综合征患者的临床结局 血红蛋白疾病，如镰状细胞病(SCD)。FDA批准的唯一治疗SCD的药物是 羟基脲，通过上调HbF发挥作用。然而，不同的患者其疗效是不同的。 而且其作用机制还不是很清楚。因此，确定上调HBF的方法，如 抑制HBF抑制子，一直是该领域的研究热点。BCL11A和LRF是转录因子 与相关的协同调节因子独立发挥作用以抑制HBF，但在治疗潜力方面存在局限性。 虽然这些转录因子及其共同调节因子已经被广泛研究，但上游调控 这些转录因子，如转录后调控，还没有得到很好的研究。澄清这些问题 未知的机制可能会发现新的治疗靶点，可以绕过针对这些目标的限制 主要的HBF抑制因子保持不变。为此，我采用了基于CRISPR/CAS9的筛选方法来询问 HBF基因调控中的RNA结合蛋白(RBP)。 使用人类红系祖细胞系HUDEP2细胞，我们询问了527个人的RBPs，发现 属于称为非均相核的一类限制性商业惯例的几个限制性商业惯例的耗竭 核糖核蛋白(HnRNP)显著上调HbF。在这些蛋白质中，效果最好的候选蛋白质 大小为突触素结合胞浆RNA相互作用蛋白(SYNCRIP)。我们通过以下方式验证了这一结果 用CRISPR/Cas9基因敲除HUDEP2细胞中的SYNCRIP并通过Flow检测HBF水平 流式细胞术、免疫印迹和RT-qPCR。我们发现，在SYNCRIP敲除后，HBF的表达是 在转录和蛋白水平上不影响BCL11A或LRF的情况下显著增加。 HnRNP是一类限制性商业惯例，对转录后调控的多个方面很重要， 如前信使核糖核酸剪接、信使核糖核酸转运、稳定和翻译等。目前，hnRNP还没有被 与HBF抑制有关，而有关SYNCRIP在造血方面的研究有限。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。 我推测，SYNCRIP与其他hnRNP一起，调节转录本的RNA加工 编码与BCL11A或LRF相关的共调控因子。在目标1中，我将研究SYNCRIP的RNA所扮演的角色 结合活性在调节HBF的表达中起作用。值得注意的是，已知的另外两个hnRNP相互调节 在这张屏幕上也发现了。因此，在目标2中，我将研究这些hnRNPs的协作机制 在HBF法规中。 通过成功完成这些目标，我将获得关于HBF这一新颖模式的更多信息 抑制，这可能被利用来治疗目的，以减轻SCD。

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.