Key Points• LIN28B regulates HbF expression in erythroblasts that are cultured from umbilical cord and adult human blood.• LIN28B expression manifested a more fetal-like phenotype among adult human erythroblasts.Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and b-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-toadult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD341 cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of g-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-toadult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of g-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype. (Blood. 2013;122(6):1034-1041 IntroductionIn humans and some other mammals, the composition of hemoglobin tetramers in erythrocytes switch from fetal hemoglobin (HbF) (a2g2) to adult hemoglobin (HbA) (a2b2) during the last stages of fetal development until early infancy.1 HbF is the most important known modifier of the clinical symptoms for patients with sickle cell disease (SCD) and b-thalassemias, which are among the most common genetic disorders worldwide. 2,3 In patients with SCD, the polymerization of sickle hemoglobin results in erythrocyte deformation and hemolysis.4 SCD patient's clinical outcomes are largely improved by inhibition of the polymerization by HbF. 5 In b-thalassemias, decreased production of b-globin causes imbalanced globin polypeptide chain synthesis, and leads to severe effects on the erythroid cells' maturation and survival. The loss of b-globin expression may be compensated by an increase in HbF production that leads to improvement of the clinical phenotype. 6 The molecular mechanisms underlying the switch from HbF to HbA are still largely unknown. Genome-wide association studies (GWAS) in both normal individuals and patients with b-hemoglobinopathies have identified BCL11A, HSB1L-MYB, and HBB clusters as having an association with the persistence of Hb...

BackgroundMicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.MethodsExpression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.ResultsAmong 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.ConclusionThese data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.

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Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34 ؉ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal,