A bipartite mechanism for IGF2BP1 in regulation of fetal hemoglobin

Abstract

Despite over a century of work on hemoglobinopathies, among them sickle cell disease, a complete understanding of hemoglobin switching and factors regulating hemoglobin expression remains incomplete even as the first CRISPR/Cas9 gene therapy has obtained approvals for the treatment of transfusion-dependent β-thalassemia and sickle cell disease. DDX6, an RNA helicase and P-body nucleator associated with miRNA-mediated RNA decay and was identified as an HbF repressor in a genome-wide CRISPR screen performed in our lab. I performed a CRISPR tiling screen on DDX6 using SpRY-ABE8e-Cas9 to achieve near base-pair resolution and identify a well-tolerated variant that induces fetal hemoglobin expression, useful to enable the further study of the role of DDX6 in regulation of fetal hemoglobin. I studied factors regulating the heterochronically silenced m6A RNA-binding protein IGF2BP1 which has been identified as a positive regulator of fetal hemoglobin expression by suppressing BCL11A expression. In contrast to this observation, post-transcriptional control by IGF2BP1 generally acts through improving the translation and mRNA stability of IGF2BP1 target mRNAs in an m6A-dependent manner. To better characterize the IGF2BP1-mediated mechanism responsible for control of fetal hemoglobin expression, I assessed whether BCL11A was required for IGF2BP1-mediated regulation of fetal hemoglobin and discovered a direct BCL11A-independent mechanism. I developed a luminescent transgenic reporter to disentangle the BCL11A¬¬-dependent and newly identified BCL11A-independent mechanisms and used it discovered that IGF2BP1 directly supports the translation of HBG mRNA in a manner requiring an intact stop codon-proximal m6A motif. In addition to this BCL11A-independent mechanism, I identified that the BCL11A-dependent mechanism relies on IGF2BP1-mediated post-transcriptional control of HIC2, which indirectly supports fetal hemoglobin expression via suppression of BCL11A. This work establishes a strategy for near-base pair resolution CRISPR base editing screening and validation of variants of interest in a novel fetal hemoglobin reporter cell line. This gene, DDX6, is likely involved in post-transcriptional control of factors regulating fetal hemoglobin. Additionally, I characterize a heretofore misunderstood BCL11A-dependent mechanism for IGF2BP1 in regulating fetal hemoglobin expression while simultaneously identifying a novel direct effect of IGF2BP1 on the fetal hemoglobin RNA, a strategy with potential to be exploited for the improved expression of lentiviral gene therapy vectors delivering HBB for sickle cell disease or transfusion dependent β-thalassemia.