Discovery of Novel EPO/EPOR/JAK2 Targets and Signal Transduction Factors Via Proteomic-Based Interrogations of Post-Translational Motif Modifications

Abstract Although EPO/EPOR/JAK2 actions have been intensely studied, the majority of investigations to date have relied heavily upon conventional approaches, extant reagents and murine cell models. We therefore hypothesized that important EPOR/JAK2 targets consequently remain undiscovered, and by applying (phospho)proteomic based interrogations, have uncovered intriguing sets of new candidate mediators of EPO’s effects on human erythroid progenitor cells. Specifically, following [-] vs [+] EPO challenge, total cellular tryptic or Glu-C peptides were generated, and those post-translationally modified (PTM) due to EPO at phosphotyrosine residues (p-Y), p-TPP motifs, and MAPK plus ATK signaling nodes were isolated, and identified by duplicate tandem LC-MS/MS. EPO targets furthermore were confirmed via parallel analyses using an EPOR agonist. Known EPO/EPOR/JAK2 targets first were validated (eg., JAK2, STAT5, GAB1-3, SHIP1-3, PLCgamma, p85alpha-PI3K, LNK, SHC1, SPRY1) (and further defined with regards to specific isoforms, plus known vs novel sites of EPO-induced PTM). Beyond this, newly discovered EPO/EPOR/JAK2 regulated targets included those within the following five categories: 1] Cytoskeletal targets unexpectedly as erythroid alpha and beta spectrins (pY2332, pY15) together with pY modulation of Beta-Adducin at a calmodulin binding site (plus EPO-regulated PTM of Calmodulin per se); 2] Select EPO- regulated transcription factors, including 100-fold p-Y modulation of KLF5 at a novel transcriptional activation motif; 3] EPO/EPOR/JAK2 modulation of two new protein tyrosine phosphatase targets as PTPN18 and PTPN7; 4] Rapid EPO-induction of a notable number of novel upstream molecular adaptors including C1ORF186/”RHEX”, C1ORF150, DOK1, IRS2, SH2D2A, STAM2, SPRY2,4, DLG1,3, FRS2, AMOTL1, RAI14, CTNND1, EPS15, HGS, ITSN2, and WASL – with two novel ORF factors as C1ORF186/RHEX and C1ORF150 as prime targets in human erythroid progenitors, but absent from rat, mouse, and lower vertebrate genomes. [For C1ORF186/RHEX, select cell and molecular biological properties will be summarized]. And 5] highlighted additional novel targets (eg, transporters, metabolic regulators) that, together with the above, further emphasize a previously underestimated sophistication of EPOR/JAK2 signal transduction circuits, certain of which are proposed to be central to EPO and ESA effects on anemia (and potentially, hypertensive side-effects). Disclosures No relevant conflicts of interest to declare.

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Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v90.2.651 ◽

1997 ◽

Vol 90 (2) ◽

pp. 651-657 ◽

Cited By ~ 41

Author(s):

Xingwei Sui ◽

Sanford B. Krantz ◽

Zhizhuang Zhao

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Gel Filtration ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Normal Cells ◽

Protein Tyrosine ◽

Erythroid Progenitor Cells

Abstract Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2021-153814 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2189-2189

Author(s):

Mark C Wilkes ◽

Aya Shibuya ◽

Vanessa M Scanlon ◽

Hee-Don Chae ◽

Anupama Narla ◽

...

Keyword(s):

Cord Blood ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Culture Media ◽

Hsp70 Expression ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

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Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽

10.1182/blood-2021-152898 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 288-288

Author(s):

Suhita Ray ◽

Linda Chee ◽

Nicholas T. Woods ◽

Kyle J Hewitt

Keyword(s):

Steady State ◽

Hemolytic Anemia ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Interacting Proteins ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Capping Protein ◽

Sam Domain ◽

Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

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Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.777.777 ◽

2009 ◽

Vol 114 (22) ◽

pp. 777-777

Author(s):

Pradeep Sathyanarayana ◽

Anamika Pradeep ◽

Jonathan D. Licht ◽

Don M. Wojchowski

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Cell Formation ◽

Strong Increase ◽

Erythroid Progenitors ◽

Kinase Activation ◽

Cellular Models ◽

Erythropoietin Treatment ◽

Molecular Adaptors

Abstract Abstract 777 The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation. Disclosures: No relevant conflicts of interest to declare.

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Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽

10.1182/blood.v128.22.2453.2453 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2453-2453

Author(s):

Susana Brito dos Santos ◽

Mark C. Allenby ◽

Athanasios Mantalaris ◽

Nicki Panoskaltsis

Keyword(s):

Mononuclear Cells ◽

Cytokine Production ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Erythroid Progenitors ◽

Variable Intensity ◽

Erythroid Progenitor ◽

Tnf Α ◽

The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

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SIRT1 Deficiency Compromises Mouse Embryonic Stem Cell Differentiation, and Embryonic and Adult Hematopoiesis In the Mouse.

Blood ◽

10.1182/blood.v116.21.1612.1612 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1612-1612

Author(s):

Xuan Ou ◽

Hee-Don Chae ◽

Rui-Hong Wang ◽

William C Shelley ◽

Scott Cooper ◽

...

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Embryonic Stem Cell ◽

Conflicts Of Interest ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor

Abstract Abstract 1612 SIRT1 is a conserved NAD-dependent deacetylase capable of deacetylating a number of protein substrates including, but not limited to, p53 and FOXO transcription factors. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). SIRT1 is expressed at high levels in mouse embryos. A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance and differentiation is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241, Calvanese V et al, PNAS, 2010; 10713736). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic progenitors (HPCs), and in embryonic and adult hematopoiesis. We hypothesized that SIRT1 is involved in hematopoietic commitment within the mouse. We initially assessed the ability of WT and SIRT1-/- mESC to give rise to blast colony forming cells (BL-CFC), a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. SIRT1-/- ESCs exhibited markedly delayed formation of BL-CFC. The emergence of the Flk-1+/c-Kit- cell population pattern was also delayed, consistent with the delayed pattern of BL-CFC development in SIRT1-/- EBs. This observed delay appears to result from a slower differentiation of the SIRT1-/- ESCs as the kinetics of decline in secondary EB potential, an indication of undifferentiated ES cells, is delayed compared to that of SIRT1+/+ ES cells. When analyzed for hematopoietic and endothelial potential of individual blast colony, replated SIRT1-/- BL-CFC presented limited hematopoietic potential, whereas endothelial potential was essentially unaltered. Next, the ability of SIRT1-/- ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1-/- R1 cells were not only delayed but greatly decreased. Moreover, after differentiation of SIRT1 -/- mESC there were also significant decreases in granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. Differences in primitive and definitive erythroid progenitors were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), a marker for primitive erythroid cells, and βmajor globin (adult hemoglobin). The above delay defects were associated with delayed ability to switch off Oct4, Nanog and Fgf5, decreased β-H1 Globin, β-major globin, Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ESC commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm SIRT1 effects, we assessed embryonic and adult hematopoiesis in SIRT1+/+, +/− and -/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythorid precursors compared to wild-type and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow HPCs in 5 week and 12 month old mice, effects especially notable at lower (5%) O2 tension. Taken together, these results demonstrate that SIRT1 plays a role in mouse embryonic and adult stem cell differentiation. Disclosures: No relevant conflicts of interest to declare.

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Effects of EGF-R on Signal Transduction and Anti-Apoptotic Cascades in Hematopoietic Cells: EGF-R Activates Multiple Signal Transduction Cascades Which Result in the Prevention of Apoptosis in the Absence of Autocrine Cytokines.

Blood ◽

10.1182/blood.v104.11.1264.1264 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1264-1264

Author(s):

John G. Shelton ◽

Linda S. Steelman ◽

Martin McMahon ◽

James A. McCubrey

Keyword(s):

Signal Transduction ◽

Hematopoietic Cells ◽

Annexin V ◽

Mtor Inhibitors ◽

Biological Properties ◽

Signal Transduction Pathways ◽

Apoptotic Response ◽

Erythroid Progenitor ◽

Multiple Signal ◽

Jnk Inhibitors

Abstract v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGF-R) that was initially discovered in the genome of avian erythoblastosis virus. v-ErbB will abrogate the requirement of erythroid progenitor cells for erythropoietin and stem cell factor and block terminal differentiation. EGF-R overexpression has been observed in many pathological situations and there is a truncated form of EGF-R referred to as EGFvIII which resembles v-ErbB in biological properties. EGF-R expression is often constitutive and may occur in the presence of expression of other oncogenes or in the absence of certain tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contained the estrogen receptor hormone binding domain which renders the v-ErbB protein dependent upon the addition of beta-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of human (TF-1) and murine (FL5.12 and FDC-P1) hematopoietic cells efficiently. This construct allowed examination of the signal transduction and anti-apoptotic pathways activated by v-ErbB:ER in hematopoietic cells. By determining which signal transduction pathways were activated in response to either v-ErbB:ER or IL-3 in the presence and absence of inhibitors, we could ascertain that v-ErbB:ER expression activated the Jak/STAT, Raf/MEK/ERK, PI3K/Akt/mTOR/p70S6K and JNK pathways. Thus v-ErbB:ER activated a broad spectrum of signal transduction pathways some of which were linked to the prevention of apoptosis. Apoptosis was measured by annexin V/PI binding and activation of caspases 3, 8 & 9. Treatment of v-ErbB:ER cells with the EGF-R inhibitor AG1478 efficiently induced apoptosis in these cells at 100 to 1000 fold lower concentrations than MEK, PI3K/mTOR or JNK inhibitors, and activation of all these signal transduction pathways was inhibited with the EGFR inhibitor. Induction of apoptosis by the EGF-R inhibitor was only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, other inhibitors suppressed kinase activation and induced some apoptosis when the cells were grown in response to v-ErbB:ER or IL-3. When the cells were treated with MEK, PI3K/mTOR or JNK inhibitors, only the kinases specific for those pathways were inhibited. When MEK and PI3K/mTOR inhibitors were added a synergistic apoptotic response was observed. In contrast, combinations of Jak or JNK and either MEK or PI3K/mTOR inhibitors did not result in a synergistic apoptotic response. Thus the Raf/MEK/ERK and PI3K/Akt/mTOR/p70S6K pathways are the most important pathways in preventing apoptosis in these cells. FL5.12 cells conditionally transformed to grow in response to activated Raf and Akt, (FL/Akt:ER+Raf-1:AR) were also isolated. Activation of STAT5 by either Raf or Akt did not occur in these cells, but did occur upon IL-3 treatment. Furthermore, Elk activation occurred in response to Raf activation but not IL-3 stimulation in the cells which grew in response to Raf and Akt. v-ErbB:ER is more effective in its ability to abrogate cytokine dependence of hematopoietic cells than Raf or Akt, and it induces multiple signal transduction pathways, only some of which are required for growth and the prevention of apoptosis in tissue culture systems.

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Genome-Wide ChIP-Seq Reveals a Dramatic Shift in the EKLF Binding Profile Between Erythroid Progenitors and Erythroblasts.

Blood ◽

10.1182/blood.v114.22.565.565 ◽

2009 ◽

Vol 114 (22) ◽

pp. 565-565 ◽

Cited By ~ 2

Author(s):

Andre M. Pilon ◽

Subramanian S. Ajay ◽

Hatice Ozel Abaan ◽

Elliott H. Margulies ◽

Patrick G. Gallagher ◽

...

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Software Package ◽

Target Genes ◽

Conflicts Of Interest ◽

Massively Parallel Sequencing ◽

Control Factor ◽

Erythroid Progenitors ◽

Binding Profile ◽

Genome Wide

Abstract Abstract 565 Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of C2H2 zinc finger transcription factors. First identified as an activator of the beta-globin locus, EKLF facilitates chromatin remodeling and transcriptional activation of target genes, at least in part through recognition of a 9-base consensus motif (NCNCNCCCN). By comparing the transcriptional profiles of E13.5 wild type and Eklf-/- mice, we demonstrated that the lethal failure to complete definitive erythropoiesis in the fetal liver (FL) was due in part to dysregulation of an EKLF target gene, the cell cycle control factor, E2F2 (Pilon et al. 2008). To identify further direct targets of EKLF activation that affect erythropoiesis, we are coupling chromatin immunoprecipitation with ultra high-throughput massively parallel sequencing (ChIP-seq). ChIP-seq is increasingly being used to map protein-DNA interactions in vivo, allowing simultaneous genome-wide analysis of transcription factor occupancy, defining an ‘interactome‘. Using mice whose endogenous Eklf gene was replaced with a fully functional HA-tagged form of EKLF, chromatin was isolated at E13.5 from immature erythroid progenitors and maturing erythroblasts by ChIP. Using a highly specific high-affinity anti-HA antibody, libraries of HA-EKLF-bound chromatin were subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signature tags that were mapped to the mouse genome using the Eland software package. A control library was derived from E13.5 FL chromatin that was not enriched for HA-EKLF occupancy. For both progenitors and erythroblasts, >1.1×107tags were obtained. 72.5% and 78.7% of progenitor and erythroblast tags mapped to unique sites within the genome, respectively. The tags were highly enriched in the ∼10% of the genome within genes (genic; 42% of tags), sites ≤10 kb from the nearest gene (adjacent; 15%), as opposed to the ∼90% of the genome that is >10 kb from the nearest gene (intergenic; 22%) or in repetitive DNA (21%) p=2.2 ×10-16. Using the MACS software package clustered peaks of EKLF occupancy were identified throughout the genome, defining the EKLF ‘interactome‘. The vast majority of peaks were mapped to non-repetitive regions of the genome (98% in progenitors; 95% in erythroblasts). Progenitors contained 4,383 peaks of EKLF occupancy, while erythroblasts contained 15,396 peaks. Only 100 peaks were common between populations. This >3.5-fold increase in genomic EKLF occupancy between progenitors and erythroblasts (p=1×10-5) reflects the shift in the expression and activity of EKLF protein in erythropoiesis described previously (Bouilloux et al. 2008; Lohmann & Bieker 2008). To identify potential EKLF target genes, we partitioned the genome into 3 categories, relative to annotated RefSeq coordinates (genic) as well as adjacent and intergenic. In progenitors, the majority of EKLF binding (54%) occurred in intergenic regions, with a minority within (38%) or adjacent (7%) to genes. By contrast, the EKLF binding profile in erythroblasts was reversed, with 62% of the peaks in genic regions, and a minority at intergenic (26%) or adjacent (12%) sites.To assess the effect of this shift in EKLF binding on gene transcription, we used publicly availabel data from the inducible G1E model of erythroid maturation (GEO: GSE628) to correlate our ChIP-seq data with mRNA expression. Informatic analyses using MetaCore demonstrated that >2,200 EKLF-associated genes were differentially expressed during maturation (949 increasing expression; 1,298 decreasing expression, all p<0.05). Among progenitors, control of cell cycle S-phase entry and progression was a significantly represented network, highlighted by focal EKLF target genes like Cdk2, Cdk4, and p107, in agreement with our previous observations. Among erythroblasts, the erythropoietin (Epo) signaling pathway was most significantly represented, highlighted by focal EKLF target genes like Stat3 and Bcl-XL, reflecting the well-established importance of the Epo axis for erythroblast survival. These data indicate that shifts in EKLF occupancy during erythropoiesis correlate with distinct functional effects on gene expression. Further, these observations support a model in which transcriptional regulators (e.g., EKLF) may collect at intergenic locations when their activity is not required, but where they remain poised for rapid recruitment. Disclosures: No relevant conflicts of interest to declare.

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PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.

Blood ◽

10.1182/blood.v116.21.2599.2599 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2599-2599

Author(s):

Ying Li ◽

Changwang Deng ◽

Xin Hu ◽

Xueqi Fu ◽

Yi Qiu ◽

...

Keyword(s):

Progenitor Cells ◽

Transcriptional Activation ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Erythroid Progenitor ◽

Functional Link ◽

Dynamic Regulation ◽

Helix Loop Helix ◽

Interacting Domain

Abstract Abstract 2599 TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains interacted with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. Disclosures: No relevant conflicts of interest to declare.

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