Direct and indirect regulation of fetal globin transcript by RNA-binding protein IGF2BP1

Despite extensive investigation, mechanisms of developmental hemoglobin expression remain incompletely understood. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N-6 methyladenosine (m6A) RNA-binding protein, IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through a poorly understood mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. Together this work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts. HUDEP-2 cells were treated with a lentiviral overexpression vector for IGF2BP1, an empty control vector, or untreated. These HUDEP-2 cells as well as wild-type HUDEP-1 cells were then differentiated for 8 days in erythroid differentiation media (EDM-II) before RNA was collected and RNAseq was paired-end RNAseq was performed with truseq adapters. Additionally, we performed RNAseq from in vitro erythroid differentiated primary cells isolated from adult bone marrow, cord blood, or fetal liver CD34+ hematopoietic stem and progenitor cells (HSPCs). Samples were quantitated using salmon and resulting counts were used for differential gene expression analysis using DEseq2.