The mRNA-Binding Protein IGF2BP1 Restores Fetal Hemoglobin in Cultured Erythroid Cells from Patients with β-Hemoglobin Disorders

Abstract

Sickle cell disease (SCD) and β-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α₂β₂), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α₂γ₂) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34⁺ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34⁺ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in β-thalassemia or potently suppress expression of sickle β-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult β-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe β-hemoglobin disorders by reversing the developmental γ- to β-globin switch.

Keywords: IGF2BP1; beta-thalassemia; fetal hemoglobin; gene regulation; gene therapy; hemoglobinopathies; lentivirus; sickle cell disease.

Graphical abstract

Figure 1

Expression and Epigenetic Analysis of IGF2BP1 in Fetal and Adult Erythroblasts (A) qRT-PCR analysis of IGF2BP1, LRF/ZBTB7A, and BCL11A mRNA in erythroblasts derived from fetal liver versus adult bone marrow CD34⁺ cells after 8 days of culture (n = 3 donors each). The expression level is reported as percentage of the internal control RNaseP and the mean ± SD is plotted. (B) ChIP sequencing results used to generate a genome-wide map of histone modifications in culture-differentiated fetal and adult erythroblasts were re-analyzed to determine epigenetic modifications for IGF2BP1. Shown are results for markers of active chromatin: H3K27 acetylation (H3K27ac), H3K4 trimethylation (H3K4me3), and H3K36 trimethylation (H3K36me3) or repressive chromatin: H3K9 trimethylation (H3K9me3) and K3K27 trimethylation (H3K27me3) in fetal (orange) and adult cells (blue). ns, not significant; *p ≤ 0.01, **p ≤ 0.001 determined by unpaired Student’s t test (two-tailed).

Figure 2

IGF2BP1 Induces a Fetal Pattern of Hemoglobin Expression in Healthy Adult Erythroblasts CD34⁺ cells from four healthy adult donors were transduced using mock conditions or with lentivirus encoding for GFP (control) or an α-spectrin regulated IGF2BP1 cassette that included 2A-ZsGreen (SI2AZG), 2A-puromycin (SI2AP), or IRES-puromycin (SIiP) and differentiated into erythroblasts. (A) qRT-PCR analysis showing the ratio of γ-globin mRNA to total (γ-globin + β-globin) mRNA plotted as mean ± SEM. (B) Percentage HbF of total hemoglobin (HbF + HbA) determined by HPLC of differentiated cell lysates for the indicated conditions plotted as mean ± SEM. (C) Representative dot plots showing expression of IGF2BP1 and HbF for mock, GFP, or IGF2BP1-transduced cells. (D) Percentage of cells double positive for IGF2BP1 and HbF plotted as mean ± SEM. *p ≤ 0.05, **p ≤ 0.001 determined by one-way ANOVA with Newman-Keuls post hoc analysis.

Figure 3

IGF2BP1 Augments HbF in β-Thalassemia Erythroblasts Bone marrow CD34⁺ cells from two adults diagnosed with β-thalassemia were transduced using mock conditions or with lentivirus encoding for GFP (control) or the IGF2BP1-2A-puromycin cassette under control of the erythroid-specific SPTA1 promoter (SI2AP) or constitutive SFFV promoter (FI2AP). Transduced cells were expanded for 7 days before samples were collected for RNA, protein, or flow cytometry and the remaining cells were placed into differentiation medium. (A) Overlay of representative flow cytometry histograms and (B) western blot demonstrating expression of IGF2BP1 for the indicated transduction conditions. Tubulin signal, shown below each lane of the western blot, served as a loading control. Molecular mass is shown in kDa. (C) qRT-PCR analysis showing the ratio of γ-globin mRNA to total (γ-globin + β-globin) mRNA for each patient. (D) Dot plots showing expression of IGF2BP1 and HbF for cells transduced with GFP control or IGF2BP1 under control of the SPTA1 (SI2AP) or SFFV (FI2AP) promoter. (E) Cellulose acetate hemoglobin electrophoresis of lysates from differentiated erythroblasts. Control samples included lysates prepared from peripheral blood of a healthy donor (PB) or umbilical cord blood (CB). M, standard consisting of sickle (HbS), fetal (HbF), and adult (HbA) hemoglobin. Percentage HbF of total hemoglobin (HbF + HbA-like [HbA₂, HbA]) determined by HPLC of differentiated cell lysates is reported below each lane. (F) Reverse-phase HPLC analysis of globin chains in differentiated cell lysates for GFP control (orange) and IGF2BP1 (purple) cell populations.

Figure 4

IGF2BP1 Reverses Defective β-Globin Expression in SCD Erythroblasts Bone marrow CD34⁺ cells from an SCD patient were transduced using conditions identical to those for the β-thalassemia patients and equivalent samples were collected. (A and B) Expression of IGF2BP1 for the indicated transduction conditions demonstrated by (A) overlay of flow cytometry histograms and (B) western blot. Tubulin signal, shown below each lane of the western blot, served as a loading control. Molecular mass is shown in kDa. (C) qRT-PCR analysis showing the ratio of γ-globin mRNA to total (γ-globin + β-globin) mRNA. (D) Cellulose acetate hemoglobin electrophoresis of lysates from differentiated erythroblasts. Control lysates were from peripheral blood of a healthy donor (PB) or umbilical cord blood (CB). M, standard consisting of sickle (HbS), fetal (HbF), and adult (HbA) hemoglobin. Percentage HbF of total hemoglobin (HbF + HbA-like [HbA₂, HbA, HbS]) determined by HPLC of differentiated cell lysates is reported below each lane.

Figure 5

IGF2BP1 Promotes Interaction between the LCR and γ-Globin Genes Cytokine-mobilized CD34⁺ cells from a healthy adult donor were transduced in triplicate with GFP control or SI2AP lentivirus particles. (A) Hemoglobin electrophoresis of differentiated cell lysates. Percentage HbF of total hemoglobin (HbF + HbA) determined by HPLC is reported below each lane. Control samples were from adult peripheral blood (PB) or umbilical cord blood (CB). M, standard consisting of sickle (HbS), fetal (HbF), and adult (HbA) hemoglobin. (B) 3C assay measuring relative crosslinking frequencies between the anchor fragment (vertical blue line) and globin genes in GFP control (orange) and IGF2BP1 (purple)-expressing cells. Each EcoRI cleavage site is represented by a red triangle, and globin genes are identified with black rectangles. Data are plotted as mean ± SD. (C) Reverse-phase HPLC analysis of globin chains in differentiated cell lysates for GFP control (orange) and IGF2BP1 (purple) cell populations. *p ≤ 0.05 determined by unpaired Student’s t test (two-tailed).