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Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.

Background In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19–40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. Methods The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. Results The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. Conclusions Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1273-x) contains supplementary material, which is available to authorized users.

Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

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., Graphical Abstract

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies.

Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.

Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult 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 CD34+ 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 γ-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-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-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.

Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.

Erythroid biology research involving rhesus macaques has been applied to several topics including malaria, hemoglobinopathy and gene therapy research. However, analyses of the rhesus red blood cells are limited by the inability to identify and sort those cells in research blood samples using flow cytometry. Here it is reported that the BRIC 6 hybridoma clone raised to the human erythroid surface molecule (referred to as CD233, Band 3, AE1, or SLC4A1) produces cross-reactive and erythroid-specific antibodies for flow cytometric detection and sorting of rhesus macaque erythrocytes.

Iron deficiency (ID) affects billions of persons worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Iron homeostasis in mammals is largely maintained by expression of a small hepatic protein named hepcidin.1 Hepcidin itself is regulated at the transcriptional and posttranscriptional levels by multiple extracellular signals related to the iron supply and the erythropoietic demands of the host, as well as by inflammatory states. ID results in a significant reduction in serum hepcidin as measured by mass spectroscopy2 and more recently by hepcidin immunoassay.3 Even in the absence of ID, anemia-related hypoxia4 and erythropoietin production inhibit hepcidin expression.5 In addition, in anemic states characterized by ineffective erythropoiesis, such as thalassemia and congenital dyserythropoiesis,6 inappropriate hepcidin down regulation results in severe iron overload. It was recently demonstrated that human erythroblasts secrete a cytokine named growth differentiation factor 15 (GDF15) that contributes to the inhibition of hepcidin in pathologic conditions.7 As a p53 target gene,8 it was proposed that GDF15 is secreted from apoptotic erythroblasts in clinical settings of ineffective erythropoiesis. However, GDF15 involvement in iron regulation in other clinical settings is largely unknown.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5′-deoxy adenosine and 2′,3′-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.

In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 +/- 50 pg/ml. In comparison, individuals with beta-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 +/- 9,600 pg/ml; range 4,800-248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

A previously undefined transcript with significant homology to the pseudo-α2 region of the α-globin locus on human chromosome 16 was detected as part of an effort to better define the transcriptional profiles of human reticulocytes. Cloning and sequencing of that transcript (GenBank AY698022; named μ-globin) revealed an insert with a 423-nucleotide open reading frame. BLASTP and ClustalW and phylogenetic analyses of the predicted protein demonstrated a high level of homology with the avian α-D globin. In addition, the heme- and globin-binding amino acids of μ-globin and avian α-D globin are largely conserved. Using quantitative real-time polymerase chain reaction (PCR), μ-globin was detected at a level of approximately 0.1% that measured for α-globin in erythroid tissues. Erythroid-specific expression was detected by Northern blot analysis, and maximal expression during the erythroblast terminal differentiation was also detected. Despite this highly regulated pattern of μ-globin gene transcription, μ-globin protein was not detected by mass spectrometry. These results suggest the human genome encodes a previously unrecognized globin member of the avian α-D family that is transcribed in a highly regulated pattern in erythroid cells. (Blood. 2005;106:1466-1472)

Here we describe the identification and characterization of an alternate delta-globin mRNA (Alt-d) discovered during high-throughput sequencing of mRNA from adult human erythroid cells. Alt-d mRNA shares the same coding region, splicing pattern, downstream untranslated region, and site of polyadenylation with the previously defined delta-globin (Delta) mRNA. Alt-d mRNA encodes an additional 145 nt in the upstream untranslated region, suggesting an alternative site of transcriptional initiation and transcription through the previously defined promoter, which contains several protein-binding motifs and a TATA box. Northern blot and PCR analyses demonstrated a restricted expression of Alt-d in fetal liver, bone marrow, and adult reticulocytes. Quantitative PCR demonstrated an Alt-d expression pattern similar to that of the Delta transcripts. In addition to intergenic RNA species and the dominant delta-globin transcripts, these data suggest that a third form of RNA is produced from low-level transcription through the delta-globin gene promoter.

Increases in fetal hemoglobin have been identified after birth in several clinical settings associated with stressed or malignant erythropoiesis. To better understand the relationship between the expression of this fetal protein and growth, donated human erythroid progenitor cells were cultured in the presence of erythropoietin (EPO) plus the growth-modifying cytokine stem cell factor (SCF), and several growth-related signaling pathways were interrogated. Only the MEK1/2 inhibitor (PD98059) demonstrated significant effects on fetal hemoglobin. In the absence of PD98059, levels of fetal hemoglobin averaged 27.4% ± 7.9% in EPO+SCF compared with 1.26% ± 1.7% in EPO alone (P = .02). A linear dose response in levels of fetal hemoglobin to PD98059 was detected (0.16 μM = 27.13%, 0.8 μM = 19.6%, 4 μM = 12.2%, 20 μM = 1.54%). Western blot analyses revealed that SCF was required for phosphorylation of MEK and p44MAPK in this setting, and quantitative polymerase chain reaction demonstrated a significant increase in γ-globin mRNA. Particular perturbations of growth-related signaling may also function to activate tissue-specific genes normally expressed during fetal development. This concept may be relevant for the development of new treatment rationales for beta hemoglobinopathies.

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)–based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Multiple components of the highly conserved let-7 cascade are developmentally regulated during the fetal-to adult transition of human ontogeny. Previous studies showed the let-7 miRNAs to be correlated with the developmentally regulated expression of fetal hemoglobin (HbF) in humans. Ex vivo manipulation of the main known let-7 regulator (LIN28) and downstream let-7 targets (IGF2BP1, IGF2BP3 and HMGA2) have shown variant increases in HbF levels (ranging from around 15-68% HbF) among cultured adult human erythroid cells. In addition, IGF2BP1 over-expression (OE) in cord blood erythroblasts raised HbF levels to an average of 90% compared to 55% in control transductions. Remarkably, in the fetal environment, the manipulation of the let-7 cascade has profound effects on HbF levels (cord blood control for the OE vectors: 57.4 ± 8.6%; LIN28A-OE: 90.8 ± 1.7%, p = 0.0003; IGF2BP3-OE: 79.0 ± 3.8%, p=0.001; HMGA2-OE: 73.4 ± 3.1, p=0.014; control for the tough decoy (TuD) vector: 51.6 ± 5.7%; let-7a-TuD: 90.3 ± 2.5%, p=0.001). As such, we hypothesized that the variable HbF effects in the adult cells may be due to independent or parallel effects from each component of the let-7 cascade since LIN28, IGF2BP1, IGF2BP3 and HMGA2 are expressed at very low levels or undetectable in the erythroid compartment after the fetal-to-adult transition. For these studies, lentiviral transduction of human CD34+ cells were investigated in erythropoietin-supplemented serum-free cultures for 21 days and compared to donor-matched control transductions. Based upon its important role in globin gene regulation, mRNA and protein expression patterns of the transcription factor BCL11A were investigated by RT-qPCR and Western blot at day 14 as a mechanism for HbF regulation by the let-7 cascade components. BCL11A mRNA was significantly down-regulated after LIN28A-OE (control: 4.1.E+03 ± 8.2.E+02 copies/ng, LIN28A-OE: 1.7.E+03 ± 1.2.E+03 copies/ng, p=0.04) and let-7a -TuD (control: 1.7E+03 ± 4.5E+02 copies/ng, let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng, p=0.003), while no significant differences were observed after IGF2BP1-OE (control: 5.42.E+03 ± 1.77.E+03 copies/ng, IGF2BP1-OE: 4.38.E+03 ± 2.57.E+02 copies/ng, p=0.375), IGF2BP3-OE (control: 5.64.E+02 ± 2.68.E+02 copies/ng, IGF2BP3-OE: 6.70.E+02 ± 3.47.E+02 copies/ng, p=0.694) and HMGA2-OE (control: 4.26E+02 ± 8.18E+01 copies/ng, HMGA2-OE: 2.84E+02 ± 1.48E+02 copies/ng, p=0.104). Interestingly, the protein levels of BCL11A were consistently down-regulated only by LIN28A-OE, let-7a -TuD and IGF2BP1-OE, with minimal changes observed after IGF2BP3-OE and HMGA2-OE. To investigate which elements of the let-7 cascade are required for the fetal hemoglobin effects in adult cells, the expression levels of each component were compared with HbF effects after lentiviral-mediated transduction of LIN28A-OE, let-7a -TuD, IGF2BP1-OE, IGF2BP3-OE, or HMGA2-OE. RT-qPCR analyses were performed at day 14 and HPLC at day 21. HbF levels for the control transductions were less than 5%. Following LIN28A-OE (HbF: LIN28A-OE: 34.8 ± 2.7%, p=0.028), both IGF2BP1 and IGF2BP3 mRNA levels remained below the detection limits, while HMGA2 was slightly down-regulated. After let-7a -TuD (HbF: let-7a-TuD: 31.0 ± 5.0%, p=0.09), LIN28A and HMGA2 remained at background levels while IGF2BP1 and IGF2BP3 were below the detection limits. Following IGF2BP1-OE (HbF: IGF2BP1-OE: 64.6 ± 3.4%, p=0.02), LIN28A, IGF2BP3, and HMGA2 remained undetectable or at background levels. After IGF2BP3-OE (HbF: IGF2BP3-OE: 22.8 ± 4.4%, p=0.021), LIN28A, IGF2BP1 and HMGA2 also remained unchanged at background or undetectable levels. Finally, after HMGA2-OE (HbF: HMGA2-OE: 13.5 ± 1.5%, p=0.01), LIN28A, IGF2BP1 and IGF2BP3 remained below the detection limits. These results were confirmed by Western analysis, with the exception of HMGA2, which was consistently increased at the protein level following LIN28A-OE, let-7a -TuD, IGF2BP1-OE and IGF2BP3-OE. In summary, these results demonstrate that several components the let-7 cascade share a downstream mechanism of BCL11A protein suppression in human erythroblasts. However, the variability of BCL11A and HbF effects as well as the ability of LIN28A-OE, let-7a-TuD and HMGA2-OE to increase HbF in the absence of IGF2BP1 and IGF2BP3 suggest both shared and independent mechanisms for hemoglobin regulation by the let-7 cascade during the human fetal-to-adult switch. Disclosures No relevant conflicts of interest to declare.

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.

OBJECTIVE: Among older children with sickle cell anemia, leukocyte counts, hemoglobin, and reticulocytosis have previously been suggested as disease severity markers. Here we explored whether these blood parameters may be useful to predict early childhood disease severity when tested in early infancy, defined as postnatal ages 60-180 days. STUDY DESIGN: Data from fifty-nine subjects who were followed at Children's National Medical Center's Sickle Cell Program for at least three years was retrospectively analyzed. Comparisons were made between white blood cell counts, hemoglobin and reticulocyte levels measured at ages 60-180 days and the clinical course of sickle cell anemia during infancy and childhood. RESULTS: A majority of subjects had demonstrable anemia with increased reticulocytosis. Only increased absolute reticulocyte levels during early infancy were associated with a significant increase in hospitalization during the first three years of life. Higher absolute reticulocyte counts were also associated with a markedly shorter time to first hospitalizations and a four-fold higher cumulative frequency of clinical manifestations over the first three years of life. No significant increase in white blood cell counts was identified among the infant subjects. CONCLUSIONS: These data suggest that during early infancy, increased reticulocytosis among asymptomatic SCA subjects is associated with increased severity of disease in childhood.

Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; >65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders. The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis. These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. Disclosures No relevant conflicts of interest to declare.

Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.

The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the Aγ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation.Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects' GDF15 levels were slightly lower than those measured in the control group of subjects (307 +/- 90 and 386 +/- 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion.ID due to blood loss is not associated with a significant change in serum levels of GDF15.

Background MicroRNAs 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. Methods Expression 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. Results Among 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. Conclusion These 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.

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, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.

Candidate drugs are being sought for the suppression of human erythropoiesis. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide] is a derivative of adenosine that inhibits the growth of leukaemic cell lines. To determine the effects of Cl-IB-MECA upon erythropoiesis, studies were performed by using an ex vivo culture system of primary human CD34+ cells. Cl-IB-MECA suppressed erythroblast growth and maturation at doses >/=50 mumol/l through a mechanism of cell cycle inhibition and accumulation of cells in the G1/G0 phase. These findings demonstrate that Cl-IB-MECA inhibits human erythropoiesis, and suggest that further consideration of this drug is warranted for patients with erythrocytosis or polycythemia syndromes.

RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009 , ARRB2, and FLJ27365 ) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.

During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Reticulocytosis begins early in life in infants with sickle cell anemia (HbSS, SCA) as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Almost one quarter of children with SCA will have at least one episode of splenic sequestration before the age of 2 years, and immature (CD36+) reticulocytes released into circulating blood may contribute to this early pathology because of splenic trapping of those cells. Additionally, clinical studies have identified that absolute reticulocyte counts (ARC) greater than 200 K/µL during early infancy (60-196 days of age) may be a useful marker to identify SCA infants who are at the highest risk for SCA-associated events during childhood. The objective of this study was to determine if expression of the adhesion marker named CD36 changes with ARC levels during early childhood in pediatric SCA patients. Infants between the ages of 6 and 12 months were enrolled in a prospective, longitudinal, observational study after written consent was obtained from a parent or legal guardian. Participants were not receiving hydroxyurea or monthly blood transfusions. After consent was provided, peripheral blood was obtained during study visits at steady state and analyzed within 48 hours of collection and storage at 40 C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated with flow cytometry following intracellular staining with a HbF fluorescent antibody. Reticulocytes were identified by flow cytometry after thiazole orange staining and were further quantitated with CD36, CD45, CD71 and CD235a staining. CD36+ reticulocytes were defined here by surface CD36 detected at levels greater than two standard deviations above unstained controls. Calculations were made to determine the proportion of total circulating reticulocytes that expressed surface CD36+ as well as the absolute number of circulating CD36+ reticulocytes/microliter of whole blood. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t-tests were used to compare means. Mean age at enrollment was 278±70 days and 62.5% of the participants were male. None of the eight enrolled patients had SCA complications or hospitalizations prior to enrollment. Mean HbF and F-cell levels were 40.8±11.1% and 92.8±8.8%, respectively. Mean hemoglobin was 10±0.6 g/dL and ARC 189.1±69.4 K/µL and mean number of absolute CD36+ red blood cells was 29.3±19.1 K/µL. ARC increased over time in 6 of the 8 (75%) participants for the study observation period. ARC was positively correlated with the proportion of CD36+ reticulocytes (r=0.78, p The incidence of splenic sequestration is highest in the first two years of life. Results from this cohort of SCA infants reveal that both ARC levels and CD36+ reticulocytes increase during the first 2 years of life, and the increases in ARC are positively correlated with the proportion and absolute number of circulating CD36+ reticulocytes in peripheral blood. This relationship provides a potential mechanism for the previously reported relationship of early reticulocytosis with increased risk of future SCA complications and events. As such, longitudinal studies should be pursued to determine if increasing CD36+ reticulocytes during early childhood serves as a marker or contributes to early SCA-related pathologies including splenic sequestration. Disclosures No relevant conflicts of interest to declare.

Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

We report here the cloning and initial characterization of a novel growth-related gene (EEG-1) that is located on the short arm of chromosome 12. Two spliced transcripts were cloned from human bone marrow and human erythroid progenitor cells: EEG-1L containing a 4350-nucleotide open reading frame encoding a putative protein of 1077 amino acids including a C1q-like globular domain, and an alternatively spliced transcript lacking exon 5 (EEG-1S) encodes a significantly smaller coding region and no C1q-like domain. Quantitative PCR revealed expression of both EEG-1 transcripts in all analyzed tissues. Plasmids encoding green fluorescent protein-tagged genes (GFP-EEG-1) were transfected into Chinese hamster ovary cells for localization and functional assays. In contrast to the diffuse cellular localization of the GFP control, GFP-EEG-1L was detected throughout the cytoplasm and excluded from the nucleus, and GFP-EEG-1S co-localized with aggregated mitochondria. Transfection of both isoforms was associated with significantly increased levels of apoptosis. Stable transfection assays additionally demonstrated decreased growth in those cells expressing EEG-1 at higher levels. Quantitative PCR analyses of mRNA obtained from differentiating erythroid cells from blood donors were performed to determine the transcriptional pattern of EEG-1 during erythropoiesis. EEG-1 expression was highly regulated with increased expression at the stage of differentiation associated with the onset of global nuclear condensation and reduced cell proliferation. We propose that the regulated expression of EEG-1 is involved in the orchestrated regulation of growth that occurs as erythroblasts shift from a highly proliferative state toward their terminal phase of differentiation.

Sir, Anaemia is a common haematological abnormality in patients with cancer. The anaemia of chronic disease or inflammation is thought to be a result of induction of the iron-regulatory hormone, hepcidin, by inflammatory cytokines, especially interleukin 6 (IL-6) [1]. Elevation of hepcidin by IL-6 leads to hypoferraemia associated with anaemia and iron-restricted erythropoiesis [2]. A recent study showed that patients with certain early-stage malignancies are less prone to developing anaemia of chronic disease [3]. Among more than 1400 men with prostate cancer, the odds of developing anaemia before diagnosis were not increased. We hypothesized that the paucity of significant anaemia during the early stages of prostate cancer may be due to potentially balanced effects of hepcidin-regulating cytokines produced by prostate cancer cells. Like IL-6, growth differentiation factor 15 (GDF15), also a cytokine, is overexpressed in prostate cancer tissues [4,5]. However, unlike IL-6, GDF15 suppresses hepcidin expression [6]. Hepcidin, IL-6 and GDF15, as well as iron and haematological variables were therefore examined to determine the potential role of iron-restricted anaemia in men with prostate cancer. In all, 29 men with prostate cancer were recruited for this preliminary study and grouped according to the presence or absence of metastatic bone disease. All studies were conducted at the National Institutes of Health (NIH) and were approved by both the Office of Human Subjects Research and the Institutional Review Board. Mild anaemia (defined here as a haemoglobin [Hb] level of 10.0–13.7 g/dL) was detected in >80% of men in both groups (Fig. 1). As expected, Hb levels were lower in men with metastatic bone disease, with a mean (SD) Hb of 11.6 (1.4) g/dL (P = 0.009) and haematocrit (Hct) of 36.5 (4.1)% (P = 0.025), than in men without metastasis, with a mean (SD) Hb of 12.8 (0.8) g/dL and Hct of 39.4 (2.1)%. None of the 29 men had received erythropoietin or transfused blood, and only one had received iron supplements within the 6 weeks prior to the study. The iron parameters were within the normal range in >80% of the men. FIG. 1 Hepcidin, IL-6, and GDF15 levels in men with prostate cancer with or with no bone metastasis. Serum concentrations of (A) hepcidin, (B) IL-6, and (C) GDF15 from men with prostate cancer, without (−, 14 men) or with (+, 15 men) bone metastasis. ... ELISA-based assay systems were used to examine the serum levels of hepcidin, GDF15 and IL-6 [6, 7]. As shown in the attached Figure 1A, 23 of 29 men studied had measurable hepcidin within the assay’s normal range (10–298 ng/mL) [7]. Five men had hepcidin levels at or near the lower limit of the assay (5.0 ng/mL). Serum hepcidin level increased beyond the normal range only in one man. In the absence of bone metastasis, hepcidin levels clustered in the low normal range, with a mean value of 42 ng/mL. Even with bone metastasis, over half of the men maintained hepcidin levels of

Globin gene expression undergoes developmental switching from embryonic (ε) through fetal (γ) to adult (δ and β) genes. Inherited mutations or deletions at the β-gene cause beta-thalassemia. One of the most propitious strategies of treatment for the disease is forced switching from mutated β-gene to unaffected fetal γ-gene expression in adult erythroid cells. Expression of globin genes is regulated by the upstream LCR enhancer. The LCR enhancer loops to globin gene promoters utilizing the LDB1/GATA-1/TAL1/LMO2 protein complex. Additionally histone-modifying enzymes play a significant role in regulation of globin gene expression. G9a methyltransferase, responsible for establishing H3K9me2 histone modification, is involved in repressing fetal and activating adult globin gene expression in mouse erythroid cells. Moreover, inhibition of G9a methyltransferase activity by the synthetic chemical compound UNC0638 activates γ- and represses β-gene expression in adult human hematopoietic precursor CD34(+) cells. Using ex vivo differentiation of primary CD34(+) adult human cells as a model system, we investigated the effect of UNC0638 on switching from β- to γ-globin gene expression, LDB1 complex occupancy and LCR/β-gene promoter looping patterns in adult erythroblast cells. Human peripheral blood CD34(+) progenitor cells from three healthy adult donors were differentiated for 21 days in a three phase serum-free media system. Based upon dose titration studies, 1µM UNC0638 was added to the medium during the most proliferative phase of culture (days 7-14) and compared to control cells grown without UNC0638. Under these conditions, a highly significant 5-fold increase in γ-globin gene expression was observed. UNC0638 treatment also caused a pronounced (3-fold) reduction in β-globin gene expression without substantial change in α-globin. At the end of the culture period, HPLC analyses also demonstrated that UNC0638 treatment resulted in a considerable increase in the cellular fetal hemoglobin (HbF / HbA + HbF: control: 2.9 +/- 1.2%; UNC0638: 30.9 +/- 2.5%, p=0.003). Chromatin immunoprecipitation and chromosome conformation capture assays were utilized to determine if the increase of fetal hemoglobin along with activation of γ-gene expression and concomitant reduction of β-gene expression were associated with epigenetic modification of the β-globin locus. UNC0638 erased H3K9me2 histone modification in the β-globin locus and caused changes in LCR looping from interaction with the β- to the γ-globin gene. Mirroring differences in looping pattern, LDB1 containing protein complex occupancy was significantly increased at the γ-globin gene and decreased at δ- and β-gene promoters. These results support a model whereby G9a establishes conditions preventing activation of γ-gene by interacting with the LCR and facilitating LCR looping with δ- and β-gene promoters and subsequent strong activation of adult globin genes expression during differentiation of adult erythroid progenitor cells. In this view, G9a inhibition represents a promising approach for treatment of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

Hemoglobin switching is largely complete in healthy infants by 6 months of age. In infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Previous studies demonstrated that patients with an ARC greater than 200 K/uL during early infancy (60-196 days of age) were at the highest risk for SCA-associated events. 1,2 The objective of this study was to determine if ARC is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study [42 (39.6%) less than 1 year of age (28-362 days old), 46 (43.4%) between the ages of 1 and 10 years, and 18 (17.0%) between 10 and 20 years old]. After consent and assent were provided, discarded peripheral blood was obtained during routine clinic visits at steady state and analyzed within 48 hours of collection and storage at 40C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated by flow cytometry following intracellular staining with a fluorescent antibody directed against HbF. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t tests were used to compare means. Initial studies compared groups based upon ARC greater than or equal to 200 K/uL (ARC≥200) during infancy because of the previously reported utility of this threshold as a predictive marker for SCA severity.1 Over one third of the infants less than 1 year of age (n=16) had an ARC≥200. Mean HbF and F-cell levels were significantly lower in the ARC≥200 group when compared to the ARC Enrolled patients were also grouped according to age and comparisons were made between ARC and HbF or F-cell levels. HbF and F-cell levels were negatively correlated to ARC in the infant subgroup (r=-0.696, p=3.1E-07 and r=-0.795, p=0.000, respectively). HbF and F-cell levels from children between the ages of 1 and 10 years were inversely related to the ARC, but the correlation was less significant (r=-0.626, p=3.3E-06 and r=-0.538, p=1.2E-04, respectively). The inverse relationship was no longer present in the oldest group of patients (HbF vs. ARC r=-0.203, p=0.420 and F-cells vs. ARC, r=-0.258, p=0.302). According to both analytical approaches described here, increased ARC is associated with decreased HbF and F-cell levels in infants with SCA. Less robust negative correlations are maintained through age 10 years, but no significant correlation was identified in adolescence and young adulthood. Overall, the data suggest that increased ARC levels may identify SCA infants who manifest a more rapid or greater loss of fetal hemoglobin during the later stages of the HbF-to-HbS switching phenomenon. Meier ER, Byrnes C, Lee YT, et al. Increased reticulocytosis during infancy is associated with increased hospitalizations in sickle cell anemia patients during the first three years of life. PLoS One 2013; 8(8):e70794. doi: 10.1371/journal.pone.0070794.Meier ER, Wright EC, Miller JL. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. Am J Hematol 2014 May 31; doi: 10.1002/ajh.23777. [Epub ahead of print] Disclosures No relevant conflicts of interest to declare.