Late Erythropoiesis Research Articles

Unveiling the genetic landscape of suspected congenital dyserythropoietic anemia type I: A retrospective cohort study of 36 patients.

Congenital Dyserythropoietic Anemia type I (CDA I) is a rare hereditary condition characterized by macrocytic/normocytic anemia, splenomegaly, iron overload, and distinct abnormalities during late erythropoiesis, particularly internuclear bridges between erythroblasts. Diagnosis of CDA I remains challenging due to its rarity, clinical heterogeneity, and overlapping phenotype with other rare hereditary anemias. In this case series, we present 36 patients with suspected CDA I. A molecular diagnosis was successfully established in 89% of cases, identifying 16 patients with CDA I through the presence of 18 causative variants in the CDAN1 or CDIN1 genes. Transcriptomic analysis of CDIN1 variants revealed impaired erythroid differentiation and disruptions in transcription, cell proliferation, and histone regulation. Conversely, 16 individuals received a different diagnosis, primarily pyruvate kinase deficiency. Comparisons between CDA I and non-CDA I patients revealed no significant differences in erythroblast morphological features. However, hemoglobin levels and red blood cell count differed between the two groups, with non-CDA I subjects being more severely affected. Notably, most patients with severe anemia belonged to the non-CDA I group (82% non-CDA I vs. 18% CDA I), with a subsequent absolute prevalence of transfusion dependency among non-CDA I patients (100% vs. 41.7%). All patients exhibited reduced bone marrow responsiveness to anemia, with a more pronounced effect observed in non-CDA I patients. Erythropoietin levels were significantly higher in non-CDA I patients compared to CDA I patients. However, evaluations of erythroferrone, soluble transferrin receptor, and hepcidin revealed no significant differences in plasma concentration between the two groups.

Hydroxyurea's Impact on Erythropoiesis, Cell Death and HbF Expression in the Hudep-2 Model

Background : Sickle cell disease (SCD) is an inherited disorder caused by a mutation in the beta-globin gene, leading to the synthesis of sickle hemoglobin called HbS. Under deoxygenated conditions, HbS polymerizes and forms fibers that weakens red blood cells (RBC) resulting in hemolysis and vaso-occlusion. Recently, ineffective erythropoiesis in SCD has been reported, characterized by higher apoptosis levels during late erythropoiesis. Interestingly, fetal hemoglobin (HbF)-expressing erythroblasts during erythroid differentiation seem to be protected from the apoptosis. Hydroxyurea (HU) is the first major approved drug for SCD in US and Europe; its proven efficacy in decreasing SCD morbidity and mortality is mainly mediated by the induction of HbF expression that is well-known as a modulator of disease severity through its ability to interfere with HbS polymerization. Despite being used for decades, HU action mechanism still remains to be fully elucidated. More precisely, HU impact on RBC maturation process is poorly documented as well as its mode of action on gamma-globin gene expression. Aim : Our aim is to investigate the effect of HU on erythropoiesis kinetics, HbF induction and apoptosis. Methods : We used HUDEP-2 (Human Umbilical cord Derived Erythroid Progenitors-2) cell line as a model for erythropoiesis. Cells were differentiated in normoxia (20% oxygen) or hypoxia (5% oxygen) - to mimic bone marrow environment, and were treated or not with HU allowing the comparison of HU effect both in normoxia and hypoxia. Analysis was performed at day D1, D3, D6, D8 and D10 of differentiation using flow cytometry, microscopy (MGG staining) and High-Performance Liquid Chromatography (HPLC). Kinetics of differentiation were followed by microscopy with MGG coloration and by flow cytometry using GPA, CD49d, Band3 and HbF antibodies. Apoptosis was assessed using annexin V staining. Results : Wedid first assess HU effect on HbF expression during erythropoiesis by flow cytometry and HPLC. Both methods showed that HU induced HbF expression from D1 to D6 of differentiation and that this induction is higher when the cells are in hypoxic conditions. We also investigated the apoptotic rate during erythropoiesis. Interestingly, HU treatment does not induce apoptosis in differentiating erythroblast, but decreased proliferation rates by 2-fold change in normoxia. Hypoxic conditions also decreased proliferative rate similarly to HU in normoxia but interestingly, in hypoxic conditions, HU did not furthermore decrease cell proliferation. Then, we investigated HU effect on the differentiation kinetics. Based on the differential expression of CD49d and Band3 along the differentiation, the percentage of each stage of differentiation (proerythroblast, basophilic, polychromatic and orthochromatic) was estimated by flow cytometry in GPA + cells at D1, D3, D6, D8 and D10 of the differentiation. Figure 1 depicted flow cytometry representative results from our 4 culture conditions at D1. Our results did show that HU accelerates HUDEP-2 differentiation as soon as D1 of culture (normoxia D1 NT vs HU: ProE 53.3% vs 44%, Baso 29.9% vs 33.1%, Poly 15.2% vs 18% and Ortho 3.3% vs 3.8%) until the end of the differentiation (D8 and 10) in normoxia. This acceleration is accentuated in hypoxic conditions (Hypoxia D1 NT vs HU: ProE 34.7% vs 17.9%, Baso 42.2% vs 50.3%, Poly 17.7% vs 25.8% and Ortho 3.8% vs 4.7%) with a cumulative effect of hypoxia and HU. All the flow cytometry results were confirmed by MGG staining and quantification by microscopy. For the same day of differentiation, HU-treated HUDEP-2 are in later stages than HU-non treated cells. Conclusion : Our study is the first to investigate the impact of HU on RBC maturation - erythropoiesis. HU treatment increases HbF expression without impacting apoptosis. The treatment tends to promote erythroid differentiation by accelerating maturation processes with stronger effect in hypoxic conditions that mimic the in vivo low oxygen environment of the bone marrow, strongly suggesting that HU has other molecular targets than inducing HbF. All these results will be confirmed in CD34 + erythroid progenitors from patients with SCD. Our results give new insights on the effect of HU on erythropoiesis that would ultimately lead to a better understanding of its mechanism of action and to the identification of new therapeutical targets to counteract SCD ineffective erythropoiesis.

Luspatercept in Combination with Recombinant-Erythropoietin in MDS RS Patients: Stimulating Early and Late Erythropoiesis

Myelodysplastic syndrome with ring sideroblasts (MDS RS) and SF3B1 mutation is generally marked by severe anemia, limited response to recombinant erythropoietin (rEPO) and consequent transfusion burden. Recently, luspatercept, a TGF-beta-pathway inhibitor, was shown beneficial in approximately 1/3 of patients by reactivating late-stage erythropoiesis. Since rEPO mainly acts on early-stage erythropoiesis, we hypothesized a possible role of combination therapy in this subset of patients. Seven patients with MDS RS treated with luspatercept in combination with rEPO at two tertiary hematologic centers in Milan, Italy were retrospectively evaluated. Luspatercept was administered according to current data sheet (1 to 1.75mg/kg every 3 weeks). Endogenous (e)EPO was evaluated before rEPO start, and patients with values < 200 IU/L received subcutaneous epoetin alpha 40,000 U/week, while those with higher values received 80,000 U/week. Hematologic data and number of RBC transfusions were collected at each cycle of luspatercept, and hematologic improvement (HI) was assessed according to the revised International Working Group (IWG) 2018. All 7 patients were males, with a median age of 83 years old (70-90). According to IPSS-R, three patients were classified as intermediate risk, while the others were lower risk. At the time of luspatercept start all patients were anemic with median Hb values of 7.3 g/dL (6.1-9.9), while median neutrophil (3.1x103/mm3, 0.8-4.9) and platelet (200x103/mm3,43-282) counts were normal. The median number of RBC units transfused in the last 8 weeks before luspatercept was 8 (3-11). Figure 1 details the timing and doses of luspatercept and rEPO treatment. In total, 5 patients received rEPO as add-on treatment due to non- or loss of response to luspatercept. Median eEPO values in these subjects were 250 (99-445) IU/L. Overall, three patients (Case 1, 2 and 3) achieved HI, and one of them became transfusion independent. Specifically, Case 1 achieved minimal HI with luspatercept after cycles 2 and 6, and lost response; rEPO was added at cycle 12 with HI after cycle 14. Case 2 initially reached HI after cycle 4 but showed several oscillations of pre-transfusion Hb (7 to 9 g/dL). At cycle 13 rEPO was added obtaining HI after cycle 20. Case 3 achieved HI after 2 cycles of luspatercept but lost response after cycle 10; rEPO was added at cycle 14 and he became transfusion independent. Patient 6 had rEPO as a pre-existing therapy and experienced a minimal HI after luspatercept start; rEPO had not been stopped to avoid a drop of pre-transfusion Hb. The other patients did not respond to luspatercept, nor to combination with rEPO, which was added at cycles 14 and 7 (Case 4 and Case 5), or already ongoing at luspatercept start in Case 7. Notably, no treatment emerging adverse events were registered with the combination. In conclusion, despite the small number of cases, these data show for the first time that combination therapy of luspatercept plus rEPO is feasible and beneficial in about one third of subjects. Concomitant stimulation of early and late-stage erythropoiesis may be an interesting strategy that requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

BackgroundHematopoiesis occurs in the bone marrow, producing a complete spectrum of blood cells to maintain homeostasis. In addition to light microscopy, chromosome analysis, and polymerase chain reaction, flow cytometry is a feasible and fast method for quantitatively analyzing hematological diseases. However, because sufficient specific cell markers are scarce, dyserythropoietic diseases are challenging to identify through flow cytometry.MethodsBone marrow samples from C57BL/B6 mice and one healthy donor were analyzed using traditional two-marker (CD71 and glycophorin A) flow cytometry analysis. After cell sorting, the gene expressions of membrane proteins in early and late erythropoiesis precursors and in nonerythroid cells were characterized using microarray analysis.ResultsAmong characterized gene candidates, aquaporin 0 (AQP0) expressed as a surface protein in early- and late-stage erythropoiesis precursors and was not expressed on nonerythroid cells. With the help of AQP0 staining, we could define up to five stages of erythropoiesis in both mouse and human bone marrow using flow cytometry. In addition, because patients with dyserythropoiesis generally exhibited a reduced population of APQ0high cells relative to healthy participants, the analysis results also suggested that the levels of APQ0high cells in early erythropoiesis serve as a novel biomarker that distinguishes normal from dysregulated erythropoiesis.ConclusionsAQP0 was successfully demonstrated to be a marker of erythroid differentiation. The expression levels of AQP0 are downregulated in patients with dyserythropoiesis, indicating a critical role of AQP0 in erythropoiesis. Accordingly, the level of AQP0high in early erythroid precursor cells may serve as a reference parameter for diagnosing diseases associated with dyserythropoiesis.

RASA3 Regulates Stage-Specific AKT Signaling and Cell Cycle Progression in Mammalian Erythropoiesis

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.

“RHEX” / C1ORF186 Governs JAK2 Activation, Differentially Modulates STAT-5 and -3, Balances Human Erythroblast Development, and Becomes Essential during Red Cell Formation

Introduction: Via phospho-PTM-targeted LC/MS proteomics, we identified RHEX as an EPO- and pY-regulated integral plasma membrane adaptor protein that associates with the h-EPOR and supports early stage human* (pro)erythroblast expansion (dexamethasone system) (JEM 211:1715-22). [*RHEX has no orthologues, is conserved in H sapiens and primates, but is not represented among mouse, rat or lower vertebrates]. Presently, by employing a 3-phase HSC-to-RBC ex vivo system, shRNA lentivirus mediated knockdowns (KD's), cytohistomorphology imaging, and molecular phenotyping (including RNA seq), we have now discovered and defined three major novel effects that RHEX exerts on erythroblast formation. As an introduction to findings: 1] RHEX supports erythroblast proliferation, balances differentiation, and is essential for the formation of maturing late-stage erythroblasts. 2] RHEX governs EPO activation of not only p-AKT, p-ERK1/2, p-STAT5 and p-STAT3 but also p-JAK2, while also inversely modulating total STAT5 and STAT3 levels. 3] RNA-seq analyses reveal that the knockdown of RHEX sharply alters factors networked within "signal transduction", "plasma membrane" and "protein binding" as top GO terms. KEGG analysis defines adhesion proteins, and oxidative factors that are strongly modulated upon RHEX KD. RHEX therefore coordinates EPO/EPOR/JAK2 signals that dynamically regulate erythroblast maturation. Results: In primary HSC- derived erythroid progenitor cells (EPCs), RHEX knockdown (KD) attenuated (pro)erythroblast proliferation up to 50% of control cultures (shNT EPCs), especially during culture phases II (SCF, EPO) and III (EPO only). During development, erythroblast phenotypes were altered in three impacting ways. During early phase-II, RHEX KD heightened the frequency of polychromatic erythroblasts (200 % over controls) (Fig panel A). During late phase-II, this increase in sh-RHEX polychromatic erythroblast frequencies persisted, while parallel control shNT erythroblasts advanced to orthochromatic erythroblasts at frequencies significantly above sh-RHEX EPCs. The orthochromatic stage therefore marks a developmental point that is first compromised due to RHEX KD. During phase-III, the late-stage development of sh-RHEX EPCs became corrupted due to RHEX KD to the extent that terminally differentiating erythroblasts became markedly mis-shapen, and failed to efficiently convert to reticulocytes and erythrocytes (Fig panel B). By d6 of phase-III, in fact, intact cells were challenging to identify in sh-RHEX EPC cultures. When EPO/EPOR/JAK2 signaling modules were analyzed, RHEX's KD in UT7epo cells dysregulated EPO- activation of not only p-ERK1/2, p-AKT, p-STAT5, p-STAT3 but also pY-JAK2 (each multi-fold). In UT7epo RHEX KD cells, total STAT5 levels were elevated, while total STAT3 levels decreased (up to10-fold). Compartmentalized signaling within membrane and cytoplasmic cell fractions was also analyzed. In control shNT cells, activated pY-JAK2 was predominantly membrane-associated. RHEX KD, however, resulted in aberrantly heightened levels of pY-JAK2 in cytoplasm. This displacement effect was also observed for activated pY-STAT5. Findings suggest that RHEX may promote the membrane association of activated pY-JAK2. In addition, RHEX KD proved to markedly limit UT7epo cell-cell aggregation. To gain insight into cellular pathways that RHEX supports, RNAseq was performed using P-I d6 erythroblasts (unaffected by RHEX KD in morphologies). For factors differentially expressed due to RHEX KD plasma membrane adhesion proteins were highly represented, with high confidence. Transcripts altered 2 to &amp;gt;10-fold, include ITGB2, ITGaX, ITGaM, ICAM3, SEL-L, CD38, CD84, PLEXIND, PANNEXIN2, ADGRG3 - and each exhibited patterned erythroid expression. Select oxidizing enzymes were also elevated in RHEX KD erythroblasts as MPO, EPX, and the NADPH oxidase subunits NCF1, NCF4 and NCF2 (50+ fold). Future investigations will assess the extent to which such factors underlie RHEX's pro-erythropoietic roles. Summary: As an EPO/EPOR/JAK2 target, RHEX governs EPO- activation of JAK2, pY-JAK2's membrane vs cytoplasmic localization, and levels of total STAT5 and STAT3. During late erythropoiesis, RHEX's actions become essential for the integrity of maturing erythroblasts. Associated mechanisms may involve RHEX's effects on cell adhesion and/or oxidation. Figure Disclosures No relevant conflicts of interest to declare.

48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p&amp;lt; 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.

Regulation of Erythroblast Protein Expression By an RNA Splicing Mechanism, Decoy Exon-Mediated Intron Retention

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells &amp;gt;5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65. Disclosures No relevant conflicts of interest to declare.

Beta3-Adrenergic Blockade Restores Erythropoietic Homeostasis in Anemia of Critical Illness

IntroductionDuring anemia of critical illness such as burn injury, erythropoietin (Epo) resistance is accompanied by impaired bone marrow (BM) erythropoiesis. The non-selective β1, β2 -adrenergic receptor (AR) blocker propranolol was effective in mitigating MafB expression in multi potential progenitors and rescuing their erythrocyte commitment in burn patients. However, peripheral HGB levels did not reflect the effect of propranolol perhaps due to defective maturation of late erythroblasts, not regulated by propranolol. Subsequently, we reported that early commitment and late maturation stages are independently orchestrated via discrete β2- and β3-AR mechanisms respectively in the BM of mice with a parallel increase in blood HGB levels. Nonetheless, it is not clear whether commitment of progenitors or maturation of erythroblasts is essential to restore erythropoietic homeostasis after burn injury. In adult mice, the spleen serves as an extra medullary erythropoietic organ under hypoxia and also functions as a primary organ of erythropoietic homeostasis (Bozzini CE et al. Am J Physiol. 1970; 219(3):724). We examined in mice, erythropoietic responses to burn injury in two organs, the spleen and BM to understand the reciprocal relationship, if any, in erythropoiesis between the two adrenergically innervated organs in response to propranolol, and selective β2- and β3- AR blockers. As variation in the size of RBCs is an indication of ineffective maturation, we also evaluated coefficient of variation of RBC size (RDW) in peripheral blood from mice. To validate our animal studies, we also measured RBC parameters in human burn patients.MethodsAnimal study: B6D2F1 mice were randomized into 2 groups, 15% TBSA (total burn surface area) scald burn and sham burn. Burn mice were given β-AR blockers or saline either via Alzet pumps eight hours after injury or by daily injections. All treatments were terminated 24 hours before harvest. Animals were euthanized to obtain spleen, femurs and blood on post burn days (PBD) as specified in results. Single cell suspensions from spleen and BM were characterized for early erythroblasts (CD71+Ter119neg), orthochromatic erythroblasts (Ter119+CD71+Syto16+), reticulocytes (Ter119+CD71+Syto16neg), erythrocytes (Ter119+), and non-erythroid cells (Ter119neg CD71neg) by flow cytometry, and expressed as 103/106 total splenocytes or total BM cells. RDW was measured using HemaTrue analyzer.Human study: Retrospective analysis was done on 19 adult burn patients enrolled in IRB approved study. All patients received standard burn care (SBC) during the study period of seven weeks. As part of another clinical trial, 9/19 patients had received propranolol along with SBC (SBC+PR). Cohorts matched for age, gender and %TBSA.ResultsAnimal study: Splenic erythropoiesis was increased after burn injury on PBD 3, 7, 14 and 21 as measured by erythrocyte and erythroblast production. Propranolol treatment for 14 days via Alzet pump alone or in combination with Epo was not sufficient to reduce splenic erythrocytes and erythroblasts that were elevated after burn injury. Between the selective blockers, only SR59320A (β3-AR) and not butoxamine (β2-AR) significantly reduced splenic erythropoiesis. This was accompanied by a decrease in splenomegaly and a reciprocal increase in BM erythropoiesis only with SR59230A treatment. RDW was significantly increased in blood after burn injury, while MCV remained within normal range. Interestingly, only SR59230A treatment and not propranolol or butoxamine reversed RDW to sham levels.Human study: Hemoglobin levels were higher at PBD 1-3 (due to hemo concentration) and decreased significantly in both groups through PBD 30-48. Mean RDW was normal at PBD 1-3 (Normal range =11.5% -14.5%) in all burn patients. Although RDW seemed to stabilize initially in SBC+PR treated patients at PBD 7-10, it increased to SBC levels by PBD 30-48. Mean corpuscular volume (MCV) remained within normal limits.ConclusionEffective maturation of late erythroblasts in BM is essential for the spleen to sense erythropoietic homeostasis, which is reestablished with β3-AR blockade after burn injury. Results emphasize only β3-AR antagonist restores RDW to sham levels and not β1/ β2-AR blocker. Increased RDW in burn patients corroborates with animal studies. Overall, this model system is suitable to study mechanisms of early and late erythropoiesis after burn injury. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis

AbstractDuring erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate–dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

Intron Retention Mechanisms That Regulate SF3B1 and Mitoferrin Gene Expression during Late Erythropoiesis

Intron retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper intronic elements, using model introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 3' splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 3' splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained introns correlate with splice site strength, but are also influenced by deeper intronic elements. For example, SLC25A28 intron 2 contains intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained introns that can either increase or decrease retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression. DisclosuresNo relevant conflicts of interest to declare.

Dysregulation of ferroportin gene expression in β(0)-thalassemia/Hb E disease.

During erythropoiesis, iron levels need to be carefully regulated to ensure there is sufficient iron available for hemoglobin synthesis, but that there is no excess to cause damage to the developing erythroblast. Iron influx to the developing erythroblast is controlled by the expression of the transferrin receptor, while iron efflux is regulated by ferroportin (FPN), the sole iron-exporting protein. FPN is encoded through multiple messenger RNAs (mRNAs) some of which contain an iron-responsive element (variant I mRNAs) and some of which do not (variant II mRNAs). This study sought to investigate the expression of the FPN mRNAs in developing erythroblasts from normal controls and β(0)-thalassemia/Hb E patients. While levels of FPN protein were relatively constant, marked reductions of the variant I message were seen in erythroblasts from β(0)-thalassemia/Hb E patients as compared to normal control cells, particularly in late erythropoiesis. Variant II mRNAs were generally increased during erythroid differentiation. No difference was seen in levels of either transferrin or ferritin heavy chain expression. While no difference was observed in labile iron pools under normal culture conditions, erythroblasts from β(0)-thalassemia/Hb E patients showed a significantly reduced expression of total FPN message under high iron conditions as compared to normal control erythroblasts. These results are consisted with dysregulation of iron efflux from the maturing erythroblast in β(0)-thalassemia/Hb E patients, and this dysregulation possibly contributes to ineffective erythropoiesis seen in these patients.

GDF11 Increased in Patients with Myelodysplastic Syndrome

Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS.Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated.Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p<0.01)and higher than that of normal controls (29.76±10.10,p<0.01); The concentration of GDF11 in low-risk MDS patients was significantly higher than that of normal controls (p<0.05). (2) The expression of CD235a in high-risk group(38.49±5.42)was not different with that in low-risk group(42.64±7.36, p>0.05). (3)In high-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET%, RBC, MCHC, Hct in peripheral blood and late erythroblast, CD235a+ cells in bone marrow(r=-0.437,r=-0.428,r=-0.444,r=-0.553,r=-0.661,r=-0.436,r=-0.52,all p<0.05),and the expression of GDF11 was positively correlated with MCV(r=0.52, p <0.05),but it was not correlated with MCH (p >0.05).(4) In low-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET% (r=-0.491Ar=-0.606,both p<0.05),it was not correlated with RBC, MCHC, MCV, MCH, Hct, late erythroblast and CD235a+ cells (all p>0.05).Conclusion GDF11 increased in patients with MDS and it was negatively correlated with late erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

All-Thiol HMGB1 Is a Critical Inducer of Anemia in Sepsis Survivors through CXCR4 Signaling

Introduction: Each year, at least 500,000 patients will survive acute sepsis. Anemia is extremely common in sepsis survivors, further interfering with quality of life and activities of daily living. The pathogenesis of anemia of sepsis is unclear. High mobility group box 1 (HMGB1) is a necessary and sufficient inductor of persistent inflammation in sepsis survivors. After translocation form the nucleus to the cytoplasm, HMGB1 undergoes epigenetic changes in response to environmental queues. Redox state is the best well understood inducer of those modifications. In experimental sepsis, the reduced all-thiol form is the predominant form throughout the first four weeks of sepsis. We recently found that HMGB1 is crucial for the development and maintenance of anemia in sepsis in part by inducing a block in differentiation of late erythropoiesis. Here we present evidence indicating a critical role of the all-thiol HMGB1 in anemia of sepsis survivors through a CXCR4-dependent mechanism.Methods and results: In order to investigate the kinetics of anemia of sepsis survivors, we first induced severe polymicrobial abdominal sepsis by surgical cecal ligation and puncture (CLP) in BALB/c mice. Sham-operated mice were used as controls. Within three days after CLP, hematocrit decreased significantly in CLP survivors three days after CLP (p≤0.001), and hemoglobin (Hb) decreased significantly within 5-7 days. Initially normocytic and normochromic, mean corpuscular hemoglobin (MCH) level was reduced significantly 15 days after CLP, while MCV dropped significantly 20 days after CLP. Sepsis-induced anemia persisted for at least 30 days. Administration of an anti-HMGB1 neutralizing monoclonal antibody (nmAb) reversed anemia. Daily administration of recombinant HMGB1 for one week was sufficient to significantly reduce Hb(p≤0.05), and MCH (p≤0.01).To better understand the role of HMGB1 during erythropoiesis, we isolated CD34+ cells from cord blood and derived them in a methylcellulose-based culture, as well as in three phase liquid culture systems. In methylcellulose, all-thiol HMGB1 (the inflammatory form secreted during necrosis) significantly reduced BFU-E formation (P≤0.0001) in a concentration-dependent way, while sulphonyl-HMGB1 (the fully oxidized form released during apoptosis) had no effect. Moreover, all-thiol HMGB1 disrupted the architecture of BFU-E colonies. In liquid culture, HMGB1 significantly reduced CD34+ cell proliferation (Fig. 1A), and reduced the proportion of GPA+ cells by flow cytometry. In the liquid culture system, all-thiol HMGB1 reduced the proportion of GPA+, CD71+ cells by D16. Recently, CXCR4 was recognized as the receptor needed for all-thiol HMGB1 signaling, after forming a heterodimer with CXCL12. In the three-phase culture system, all-thiol HMGB1 significantly reduced the proportion of GPA+, CD71+ cells, and a similar level of inhibition was found when interfering with an anti-CXCR4 nmAb, CXCR4 by adding CXCL14 (a competitive inhibitor of CXCL12), or a small molecule (AMD3100) (Fig. 1B). In the presence of HMGB1, adding CXCL12 failed to rescue erythropoiesis.Conclusions: Our findings suggest that all-thiol HMGB1, the redox form present during the first weeks after sepsis onset, has a critical role in initiating and sustaining anemia in sepsis survivors. Further, all-thiol HMGB1 interferes with late erythropoiesis through a CXCR4-dependent mechanism. This has potential therapeutic implications for a persistent disorder for which the usual support –including erythropoietin and transfusions- fails to improve outcome, survival, or both. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored.Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets.Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members.Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Trim58 Degrades Dynein and Regulates Terminal Erythropoiesis

TRIM58 is an E3 ubiquitin ligase superfamily member implicated by genome-wide association studies to regulate human erythrocyte traits. Here, we show that Trim58 expression is induced during late erythropoiesis and that its depletion by small hairpin RNAs (shRNAs) inhibits the maturation of late-stage nucleated erythroblasts to anucleate reticulocytes. Imaging flow cytometry studies demonstrate that Trim58 regulates polarization and/or extrusion of erythroblast nuclei. In vitro, Trim58 directly binds and ubiquitinates the intermediate chain of the microtubule motor dynein. In cells, Trim58 stimulates proteasome-dependent degradation of the dynein holoprotein complex. During erythropoiesis, Trim58 expression, dynein loss, and enucleation occur concomitantly, and all are inhibited by Trim58 shRNAs. Dynein regulates nuclear positioning and microtubule organization, both of which undergo dramatic changes during erythroblast enucleation. Thus, we propose that Trim58 promotes this process by eliminating dynein. Our findings identify an erythroid-specific regulator of enucleation and elucidate a previously unrecognized mechanism for controlling dynein activity.

Renal anaemia and EPO hyporesponsiveness associated with vitamin D deficiency: the potential role of inflammation

Resistance to erythropoiesis-stimulating agents (ESAs) has been observed in a considerable proportion of patients with chronic kidney disease (CKD) and it is reportedly associated with adverse outcomes, such as increased cardiovascular morbidity, faster progression to end-stage renal disease (ESRD) and all-cause mortality. The major causes of ESA resistance include chronic inflammation producing suppressive cytokines of early erythroid progenitor proliferation. In addition, pro-inflammatory cytokines stimulate hepcidin synthesis thus reducing iron availability for late erythropoiesis. Recent studies showing an association in deficiencies of the vitamin D axis with low haemoglobin (Hb) levels and ESA resistance suggest a new pathophysiological co-factor of renal anaemia. The administration of either native or active vitamin D has been associated with an improvement of anaemia and reduction in ESA requirements. Notably, these effects are not related to parathyroid hormone (PTH) values and seem to be independent on PTH suppression. Another possible explanation may be that calcitriol directly stimulates erythroid progenitors; however, this proliferative effect by extra-renal activation of 1α-hydroxylase enzyme is only a hypothesis. The majority of studies concerning vitamin D deficiency or supplementation, and degree of renal anaemia, point out the prevalent role of inflammation in the mechanism underlying these associations. Immune cells express the vitamin D receptor (VDR) which in turn is involved in the modulation of innate and adaptive immunity. VDR activation inhibits the expression of inflammatory cytokines in stromal and accessory cells and up-regulates the lymphocytic release of interleukin-10 (IL-10) exerting both anti-inflammatory activity and proliferative effects on erythroid progenitors. In CKD patients, vitamin D deficiency may stimulate immune cells within the bone marrow micro-environment to produce cytokines, inducing impaired erythropoiesis. Immune activation involves the reticuloendothelial system, increasing hepcidin synthesis and functional iron deficiency. Consequences of this inflammatory cascade are erythropoietin (EPO) resistance and anaemia. Given the key role of inflammation in the response to EPO, the therapeutic use of agents with anti-cytokines properties, such as vitamin D and paricalcitol, may provide benefit in the prevention/treatment of ESA hyporesponsiveness.

Abundance of Alternative Splicing Events and Differentiation Stage-Specific Changes in Splicing Suggest A Major Role in Regulation of Gene Expression During Late Erythropoiesis

Abstract 978Alternative pre-mRNA splicing plays a major role in development and differentiation by re-modeling the transcriptome to generate mRNAs that encode the biologically appropriate cell type-specific proteome. Earlier studies employing RT-PCR and exon microarrays demonstrated a small number of splicing changes during erythroid differentiation, one of which (in protein 4.1R) is critical for mechanical stability of the membrane skeleton. Here we report that the landscape of splicing changes executed during terminal erythropoiesis is far more extensive and highly dynamic, ultimately affecting the expression of many more proteins than previously recognized. Highly purified populations of FACS-sorted cells representing erythroblasts at distinct stages during terminal erythroid differentiation from proerythroblasts to orthochromatic erythroblasts of both human and mouse origin were used as the source of RNA for RNA-seq analysis. In total, hundreds of millions of sequence reads were obtained from three biological replicates for four (mouse) or five (human) cell populations, and reads were aligned to the Ensembl-annotated transcriptome using the Bowtie aligner. Transcript-level estimates were obtained using the streaming transcript abundance estimation tool, eXpress, expression of individual exons in “exon-inclusion” isoforms relative to total isoforms was represented as Ψ (psi), or percent spliced in, and statistical significance estimates adjusted for multiple comparisons by the Benjamini-Hochberg method. Thousands of alternative splicing events were predicted in genes with diverse functions in transcription, RNA processing, protein synthesis, membrane receptors, cytoskeletal structure, etc. Initial RT-PCR studies indicate that a high proportion of predicted alternative splicing events can be validated. Comparison of Ψ values across the differentiation series revealed that hundreds of alternative exons in erythroid transcripts exhibit substantial differences in splicing efficiency between proerythroblasts and orthrochromatic erythroblasts (ΔΨ>20%), suggesting that their splicing efficiency is regulated. Both increases and decreases in exon splicing efficiency were observed, indicating that multiple splicing regulatory pathways are active and that both splicing enhancer and splicing silencer factors are involved in the regulation. Interestingly, some of the splicing switches introduce premature translation termination codons, leading us to hypothesize that splicing-coupled nonsense mediated decay may down-regulate expression of a class of erythroid transcripts. To begin exploring mechanisms that regulate the late erythroid alternative splicing program, we used the RNA-seq data to derive differentiation stage-specific expression profiles of known splicing factors. Major changes in the expression profile of many splicing regulators were observed. hnRNPA1 was strongly down-regulated in late erythroblasts, in concert with up-regulation of the protein 4.1R splicing switch it has been shown to inhibit. Because many other exons are up-regulated with similar kinetics, hnRNPA1 may be a general inhibitor of alternative splicing in early erythroblasts. In contrast, RNA-seq data indicate that several other splicing factors including MBNL1, a known splicing factor in muscle and brain whose activity is disturbed in myotonic dystrophy, are substantially up-regulated in late erythropoiesis. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts, in conjunction with the better studied transcriptional program, plays a major role in regulating gene expression to insure synthesis of the appropriate constellation of proteins both quantitatively and qualitatively as the cells are remodeled in preparation for production of mature red cells. Disclosures:No relevant conflicts of interest to declare.

Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.

Abstract 4036Poster Board III-972The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions. Disclosures:No relevant conflicts of interest to declare.

Chromosome looping at the human α-globin locus is mediated via the major upstream regulatory element (HS −40)

AbstractPrevious studies in the mouse have shown that high levels of α-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human α cluster and their interactions with the α-globin promoter. All of these elements interact with the α-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human α globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS −40) results in loss of all the interactions and α-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human α-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.