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1.  Phosphorylation of C/EBPα Inhibits Granulopoiesis 
Molecular and Cellular Biology  2004;24(2):675-686.
CCAAT/enhancer-binding protein α (C/EBPα) is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBPα is known to induce granulopoiesis while suppressing monocyte differentiation, it is unclear how C/EBPα regulates this cell fate choice at the mechanistic level. Here we report that inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBPα. This inhibition is mediated by extracellular signal-regulated kinases 1 and/or 2 (ERK1/2), which interact with C/EBPα through an FXFP docking site and phosphorylate serine 21. As a consequence of C/EBPα phosphorylation, induction of granulocyte differentiation by C/EBPα or retinoic acid is inhibited. Our analysis of C/EBPα by fluorescent resonance energy transfer revealed that phosphorylation induces conformational changes in C/EBPα, increasing the distance between the amino termini of C/EBPα dimers. Thus, myeloid development is partly regulated by an ERK1/2-mediated change in the conformation of C/EBPα that favors monocyte differentiation by blocking granulopoiesis.
PMCID: PMC343788  PMID: 14701740
2.  CCAAT/Enhancer Binding Protein α Is a Regulatory Switch Sufficient for Induction of Granulocytic Development from Bipotential Myeloid Progenitors 
Molecular and Cellular Biology  1998;18(7):4301-4314.
The transcription factor CCAAT/enhancer binding protein α (C/EBPα) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBPα in human granulopoiesis, we studied its expression and function in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. We show that the expression of C/EBPα initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBPα alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colony-stimulating factor receptor and secondary granule protein genes. Moreover, induced expression of C/EBPα in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBPα serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes.
PMCID: PMC109014  PMID: 9632814
3.  Cyclic AMP Responsive Element Binding Proteins Are Involved in ‘Emergency’ Granulopoiesis through the Upregulation of CCAAT/Enhancer Binding Protein β 
PLoS ONE  2013;8(1):e54862.
In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPβ. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPβ-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPβ were involved in the positive regulation of C/EBPβ transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPβ mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPβ transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPβ upregulation.
PMCID: PMC3559830  PMID: 23382991
4.  Antagonism of FOG-1 and GATA factors in fate choice for the mast cell lineage 
The zinc finger transcription factor GATA-1 requires direct physical interaction with the cofactor friend of GATA-1 (FOG-1) for its essential role in erythroid and megakaryocytic development. We show that in the mast cell lineage, GATA-1 functions completely independent of FOG proteins. Moreover, we demonstrate that FOG-1 antagonizes the fate choice of multipotential progenitor cells for the mast cell lineage, and that its down-regulation is a prerequisite for mast cell development. Remarkably, ectopic expression of FOG-1 in committed mast cell progenitors redirects them into the erythroid, megakaryocytic, and granulocytic lineages. These lineage switches correlate with transcriptional down-regulation of GATA-2, an essential mast cell GATA factor, via switching of GATA-1 for GATA-2 at a key enhancer element upstream of the GATA-2 gene. These findings illustrate combinatorial control of cell fate identity by a transcription factor and its cofactor, and highlight the role of transcriptional networks in lineage determination. They also provide evidence for lineage instability during early stages of hematopoietic lineage commitment.
PMCID: PMC2275384  PMID: 18299398
5.  Identification of eosinophil lineage–committed progenitors in the murine bone marrow 
The Journal of Experimental Medicine  2005;201(12):1891-1897.
Eosinophil lineage–committed progenitors (EoPs) are phenotypically isolatable in the steady-state murine bone marrow. Purified granulocyte/monocyte progenitors (GMPs) gave rise to eosinophils as well as neutrophils and monocytes at the single cell level. Within the short-term culture of GMPs, the eosinophil potential was found exclusively in cells activating the transgenic reporter for GATA-1, a transcription factor capable of instructing eosinophil lineage commitment. These GATA-1–activating cells possessed an IL-5Rα+CD34+c-Kitlo phenotype. Normal bone marrow cells also contained IL-5Rα+CD34+c-Kitlo EoPs that gave rise exclusively to eosinophils. EoPs significantly increased in number in response to helminth infection, suggesting that the EoP stage is physiologically involved in eosinophil production in vivo. EoPs expressed eosinophil-related genes, such as the eosinophil peroxidase and the major basic protein, but did not express basophil/mast cell–related mast cell proteases. The enforced retroviral expression of IL-5Rα in GMPs did not enhance the frequency of eosinophil lineage read-outs, whereas IL-5Rα+ GMPs displayed normal neutrophil/monocyte differentiation in the presence of IL-5 alone. Thus, IL-5Rα might be expressed specifically at the EoP stage as a result of commitment into the eosinophil lineage. The newly identified EoPs could be the cellular target in the treatment of a variety of disorders mediated by eosinophils.
PMCID: PMC2212039  PMID: 15955840
6.  Inflammation Triggers Emergency Granulopoiesis through a Density-Dependent Feedback Mechanism 
PLoS ONE  2011;6(5):e19957.
Normally, neutrophil pools are maintained by homeostatic mechanisms that require the transcription factor C/EBPα. Inflammation, however, induces neutrophilia through a distinct pathway of “emergency” granulopoiesis that is dependent on C/EBPβ. Here, we show in mice that alum triggers emergency granulopoiesis through the IL-1RI-dependent induction of G-CSF. G-CSF/G-CSF-R neutralization impairs proliferative responses of hematopoietic stem and progenitor cells (HSPC) to alum, but also abrogates the acute mobilization of BM neutrophils, raising the possibility that HSPC responses to inflammation are an indirect result of the exhaustion of BM neutrophil stores. The induction of neutropenia, via depletion with Gr-1 mAb or myeloid-specific ablation of Mcl-1, elicits G-CSF via an IL-1RI-independent pathway, stimulating granulopoietic responses indistinguishable from those induced by adjuvant. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC to alum or neutropenia, but plays a role in terminal neutrophil differentiation during granulopoietic recovery. We conclude that alum elicits a transient increase in G-CSF production via IL-1RI for the mobilization of BM neutrophils, but density-dependent feedback sustains G-CSF for accelerated granulopoiesis.
PMCID: PMC3104996  PMID: 21655273
7.  Upregulation of Interleukin 6 and Granulocyte Colony-Stimulating Factor Receptors by Transcription Factor CCAAT Enhancer Binding Protein α (C/EBPα) Is Critical for Granulopoiesis  
The Journal of Experimental Medicine  1998;188(6):1173-1184.
Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein α (C/EBPα)-deficient mice. This phenotype is distinct from that of G-CSF receptor−/− mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPα. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6–responsive colony-forming units (CFU-IL6) in C/EBPα−/− mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPα-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPα target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPα-deficient mice.
PMCID: PMC2212540  PMID: 9743535
CCAAT enhancer binding protein; knockout mice; colony-forming unit; hematopoiesis; myelopoiesis
8.  Increased Fanconi C expression contributes to the emergency granulopoiesis response 
The Journal of Clinical Investigation  2013;123(9):3952-3966.
Emergency granulopoiesis is a component of the innate immune response that is induced in response to infectious or inflammatory challenge. It is characterized by the rapid expansion and differentiation of granulocyte/monocyte progenitor (GMP) populations, which is due in part to a shortened S-phase of the cell cycle. We found that IRF8 (also known as ICSBP), an interferon regulatory transcription factor that activates phagocyte effector genes during the innate immune response, activates the gene encoding Fanconi C (Fancc) in murine myeloid progenitor cells. Moreover, IRF8-induced Fancc transcription was augmented by treatment with IL-1β, an essential cytokine for emergency granulopoiesis. The Fanconi pathway participates in repair of stalled or collapsed replication forks during DNA replication, leading us to hypothesize that the Fanconi pathway contributes to genomic stability during emergency granulopoiesis. In support of this hypothesis, Fancc–/– mice developed anemia and neutropenia during repeated, failed episodes of emergency granulopoiesis. Failed emergency granulopoiesis in Fancc–/– mice was associated with excess apoptosis of HSCs and progenitor cells in the bone marrow and impaired HSC function. These studies have implications for understanding the pathogenesis of bone marrow failure in Fanconi anemia and suggest possible therapeutic approaches.
PMCID: PMC3754263  PMID: 23925293
9.  The Lineage-c-kit+Sca-1+ Cell Response to Escherichia Coli Bacteremia in Balb/c Mice 
Stem cells (Dayton, Ohio)  2008;26(7):1778-1786.
During bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for host defenses. Along with this enhancement of granulopoiesis, the bone marrow also increases its release of hematopoietic precursors. At the present time, little is known about the commitment of hematopoietic precursor cells including hematopoietic stem cells and progenitors in this response. To investigate the hematopoietic precursor cell response to bacterial infection, bacteremia was established in Balb/c mice by intravenous injection of Escherichia coli. Bacteremia caused a 10-fold increase in the number of lineage (lin)-c-kit+Sca-1+ cells in the bone marrow. This dramatic expansion of the lin-c-kit+Sca-1+ cell pool resulted from both increased mitosis of these cells and inversion from lin-c-kit+Sca-1- cell phenotype. Lipopolysaccharide, tumor necrosis factor-α, and interleukin-6 were potent factors capable of mediating phenotypic inversion of lin-c-kit+Sca-1- cells. Cells in the expanded lin-c-kit+Sca-1+ cell pool contained an increased number of colony-forming unit-granulocyte/macrophage (CFU-GM). Mobilization of lin-c-kit+Sca-1+ cells into the circulation was significantly enhanced following bacteremia. These results demonstrate that the lin-c-kit+Sca-1+ cell population in the bone marrow constitutes a key component of the host defense response to bacteremia. Functional modifications of these primitive hematopoietic precursors are critical for enhancing granulocyte production following bacterial infection.
PMCID: PMC2731662  PMID: 18483422
Mouse; Stem/progenitor Cells; Granulopoiesis; Hematopoiesis; Bone Marrow; Immunity
10.  Repression of Transcriptional Activity of C/EBPα by E2F-Dimerization Partner Complexes▿†  
Molecular and Cellular Biology  2010;30(9):2293-2304.
The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPα transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPα BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPα mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPα interacts with the dimerization partner (DP) of E2F and that C/EBPα-E2F/DP interaction prevents both binding of C/EBPα to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPα, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.
PMCID: PMC2863587  PMID: 20176812
11.  Twist-2 Controls Myeloid Lineage Development and Function 
PLoS Biology  2008;6(12):e316.
Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2–deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPα. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-γ (IFNγ) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.
Author Summary
Hematopoiesis is coordinated by transcription factors that regulate proliferation, differentiation, and cell fate determinations. Myelopoiesis refers to the development of all white blood cells, excluding lymphocytes (B and T cells); however, the molecular regulation of this developmental process is still incompletely understood. In this study using mice that lack expression of Twist-2, we establish a novel role for this basic helix-loop-helix transcription factor as regulator of myeloid progenitors and fully differentiated myeloid cells. Specifically, Twist-2 acts to inhibit proliferation as well as differentiation of progenitors that give rise to macrophages, neutrophils, and basophils by inhibiting the important transcription factors Runx1 and C/EBPα. In mature myeloid cells, Twist-2 negatively regulates the production of proinflammatory cytokines while positively promoting the production of regulatory cytokine IL-10 by these cells. These findings provide significant insight into regulation of myeloid lineage development and function.
The transcription factor Twist-2 is a new regulator that inhibits the proliferation and differentiation of granulocyte macrophage progenitors. Twist-2 also inhibits proinflammatory cytokine production, while stimulating IL-10 by myeloid cells.
PMCID: PMC2602725  PMID: 19090621
12.  C/EBPα Induces PU.1 and Interacts with AP-1 and NF-κB to Regulate Myeloid Development 
Blood cells, molecules & diseases  2007;39(3):340-343.
C/EBPα and PU.1 are key regulators of early myeloid development. Mice lacking C/EBPα or PU.1 have reduced granulocytes and monocytes. Consistent with a model in which induction of PU.1 by C/EBPα contributes to monocyte lineage specification, mice with reduced PU.1 have diminished monocytes but retain granulocytes, C/EBPα directly activates PU.1 gene transcription, and exogenous C/EBPα increases monocytic lineage commitment from bipotential myeloid progenitors. In addition to C/EBPα, AP-1 proteins also have the capacity to induce monocytic maturation. C/EBPα:c-Jun or C/EBPα:c-Fos leucine zipper heterodimers induce monopoiesis more potently than C/EBPα or c-Jun homodimers or c-Fos:c-Jun heterodimers. C/EBPs and NF-κB cooperatively regulate numerous genes during the inflammatory response. The C/EBPα basic region interacts with NF-κB p50, but not p65, to induce bcl-2, and this interaction may be relevant to myeloid cell survival and development.
PMCID: PMC2083642  PMID: 17669672
C/EBPα; PU.1; c-Jun; NF-κB; myeloid
13.  A critical role for TCF-1 in T-lineage specification and differentiation 
Nature  2011;476(7358):63-68.
The vertebrate thymus provides an inductive environment for T-cell development. Within the thymus, Notch signals are indispensable for imposing the T-cell fate on multipotential hematopoietic progenitors, but the downstream effectors that impart T-lineage specification and commitment are not well understood. Here we show that transcription factor, T-cell factor 1 (TCF-1), is a critical regulator in T-cell specification. TCF-1 is highly expressed in the earliest thymic progenitors, and its expression is upregulated by Notch signals. Most importantly, when TCF-1 is forcibly expressed in BM progenitors, it drives the development of T-lineage cells in the absence of T-inductive Notch1 signals. Further characterization of these TCF-1-induced cells revealed expression of many T-lineage genes, including T-cell specific transcription factors Gata3, Bcl11b, and components of the T-cell receptor. Our data suggest a model where Notch signals induce TCF-1, and TCF-1 in turn imprints the T-cell fate by upregulating expression of T-cell essential genes.
PMCID: PMC3156435  PMID: 21814277
14.  Sumoylation and the function of CCAAT enhancer binding protein alpha (C/EBPα̤) 
CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and is a master regulator of granulopoiesis. It is expressed at high levels throughout myeloid differentiation and binds to the promoters of multiple myeloid- specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities occur in mice nullizygous for C/EBPα̤ including a selective early block in the differentiation of granulocytes. Mutations in C/EBPα are present in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform, which contributes to loss of C/EBPα function. The molecular basis for this observation remains unknown. In addition to phoshorylation, C/EBPα is modified, post-translationally by a small ubiquitin-related modifier (SUMO) at a lysine residue (K159), which lies within the growth inhibitory region of the C/EBPα protein. Sumoylation at K159 in the C/EBPα protein prevents association of the SWI/SNF chromatin remodeling complex with C/EBPα, thereby hampering transactivation. In this review, the functional implications of post-translational modification, particularly sumoylation, of C/EBPα in normal granulopoiesis and leukemia are considered.
PMCID: PMC2505045  PMID: 18406180
transcription factor; myeloid leukemia; post-translational modification
15.  Loss of C/EBPα cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage 
CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBPα to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations—all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count—normally associated with AML—were absent. These results show that disrupting the cell cycle regulatory function of C/EBPα is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML.
PMCID: PMC2212897  PMID: 15983063
16.  C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia 
The Journal of Clinical Investigation  2012;122(12):4490-4504.
C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.
PMCID: PMC3533560  PMID: 23160200
17.  IL-1RI dependent HSC proliferation is necessary for Inflammatory Granulopoiesis and Reactive Neutrophilia1 
Infections and inflammation trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as emergency granulopoiesis. The intrinsic factors that drive reactive neutrophilias and emergency granulopoiesis have been inferred but not demonstrated. Here we show that alum can not elicit reactive neutrophilias in IL-1RI−/− mice whereas other inflammatory responses, including eosinophilia and antibody production, remain intact. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Indeed, HSC and MPP proliferative responses were most suppressed in IL-1RI−/− mice, suggesting a critical role for their proliferation in inflammatory granulopoiesis. Whereas IL-1 drives increased HSC proliferation directly in vitro, IL-1RI expression by radiation-resistant host cells was both necessary and sufficient for alum-induced HSC, MPP, and GMP proliferation and reactive neutrophilias in radiation chimeric mice. Thus, IL-1 plays a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.
PMCID: PMC2780360  PMID: 19414802
Cell Differentiation; Cytokine Receptors; Hematopoiesis; Inflammation; Neutrophils
18.  Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration 
Cell  2011;147(3):577-589.
BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.
PMCID: PMC3219441  PMID: 22036566
19.  Targeted Deletion of a High-Affinity GATA-binding Site in the GATA-1 Promoter Leads to Selective Loss of the Eosinophil Lineage In Vivo 
The Journal of Experimental Medicine  2002;195(11):1387-1395.
Transcription factor GATA-1 reprograms immature myeloid cells to three different hematopoietic lineages-erythroid cells, megakaryocytes, and eosinophils. GATA-1 is essential for maturation of erythroid and megakaryocytic precursors, as revealed by gene targeting in mice. Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss of the eosinophil lineage. These findings suggest that GATA-1 is required for specification of this lineage during hematopoietic development. Mice lacking the ability to produce eosinophils should prove useful in ascertaining the role of eosinophils in a variety of inflammatory or allergic disorders.
PMCID: PMC2193547  PMID: 12045237
eosinophil; GATA-1; gene targeting; hematopoiesis; transcription
20.  Th17 cells and Mucosal Host Defense 
Seminars in immunology  2007;19(6):377-382.
Th17 cells are a new lineage of T-cells that are controlled by the transcription factor RORγt and develop independent of GATA-3, T-bet, Stat 4 and Stat 6. Novel effector molecules produced by these cells include IL-17A, IL-17F, IL-22, and IL-26. IL-17RA binds IL-17A and IL-17F and is critical for host defense against extracellular planktonic bacteria by regulating chemokine gradients for neutrophil emigration into infected tissue sites as well as host granulopoiesis. Moreover IL-17 and IL-22 regulate the production of antimicrobial proteins in mucosal epithelium. Although TGF-β1 and IL-6 have been shown to be critical for development of Th17 cells from naïve precursors, IL-23 is also important in regulating IL-17 release in mucosal tissues in response to infectious stimuli. Compared to Th1 cells, IL-23 and IL-17 show limited roles in controlling host defense against primary infections with intracellular bacteria such as Mycobacterium tuberculosis suggesting a predominate role of the Th17 lineage in host defense against extracellular pathogens. However in the setting of chronic biofilm infections, as that occurs with Cystic Fibrosis or bronchetctasis, Th17 cells may be key contributors of tissue injury.
PMCID: PMC2293278  PMID: 18054248
21.  Carcinoembryonic antigen-related cell adhesion molecule-1 regulates granulopoiesis by inhibition of granulocyte-colony stimulating factor receptor 
Immunity  2010;33(4):620-631.
Although carcinoembryonic antigen-related cell adhesion moclecule-1 (CEACAM1) is an activation marker for neutrophils and delays neutrophil apoptosis, the role of CEACAM1 in granulopoiesis and neutrophil dependent host immune responses has not been investigated. CEACAM1 expression correlated with granulocytic differentiation, and Ceacam1−/− mice developed neutrophilia due to loss of the Src- homology-phosphatase-1 (SHP-1) dependent inhibition of granulocyte-colony stimulating factor receptor (G-CSFR)-signal transducer and activator of transcription (Stat3) pathway provided by CEACAM1. Moreover, Ceacam1−/− mice were hypersensitive to Listeria Monocytogenes (LM) infection with an accelerated mortality. Reintroduction of CEACAM1 into Ceacam1−/− bone marrow restored normal granulopoiesis and host sensitivity to LM infection, while mutation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) abrogated this restoration. shRNA mediated reduction of Stat3 amounts rescued normal granulopoiesis attenuating host sensitivity to LM infection in Ceacam1−/− mice. Thus, CEACAM1 acted as a co-inhibitory receptor for G-CSFR regulating granulopoiesis and host innate immune response to bacterial infections.
PMCID: PMC3765078  PMID: 21029969
22.  Essential and Instructive Roles of GATA Factors in Eosinophil Development 
The Journal of Experimental Medicine  2002;195(11):1379-1386.
GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.
PMCID: PMC2193540  PMID: 12045236
GATA-1; GATA-2; eosinophil; myeloid cell; allergy
23.  From hematopoietic progenitors to B cells: mechanisms of lineage restriction and commitment 
Current opinion in immunology  2010;22(2):177-184.
The generation of B lymphocytes from hematopoietic progenitors requires lineage-specific transcription factors that progressively direct cell fate choices. Differentiation of hematopoietic stem cells to lymphoid progenitors requires Ikaros-dependent lineage priming and graded levels of PU.1, which are controlled by Ikaros and Gfi1. E2A drives expression of EBF1, which initiates B lineage specification. EBF1, in addition to Pax5, is involved in commitment to the B cell lineage. As a model of gene activation in early B lymphopoiesis, mb-1 genes are activated sequentially by factors (e.g. EBF1) that initiate chromatin modifications prior to transcription. This review highlights the requisite interplay between transcription factors and epigenetic mechanisms in the context of B cell development.
PMCID: PMC2854230  PMID: 20207529
24.  Phosphorylation of Serine 248 of C/EBPα Is Dispensable for Myelopoiesis but Its Disruption Leads to a Low Penetrant Myeloid Disorder with Long Latency 
PLoS ONE  2012;7(6):e38841.
Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro.
Methodology/Principal Findings
Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of CebpaS248A/S248A knock-in mice. Surprisingly, 8-week old CebpaS248A/S248A mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from CebpaS248A/S248A animals display a competitive advantage compared to wild type cells in a transplantation assay.
Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context.
PMCID: PMC3371045  PMID: 22715416
25.  Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation 
Experimental hematology  2008;36(7):786-798.
Granulocyte colony stimulating factor (G-CSF) and interleukin-6 (IL-6) play key roles in regulating emergency granulopoiesis and inflammation, and are both negatively regulated by the inducible intracellular protein suppressor of cytokine signaling-3 (Socs3). Mice with Socs3 deleted specifically in hematopoietic cells succumb to severe neutrophil and macrophage driven inflammation by one year of age, and responses to G-CSF are grossly exacerbated. In order to determine which elements of cellular responses to cytokines require Socs3, we have examined the differentiative and proliferative capacity of hematopoietic progenitor cells stimulated by G-CSF and IL-6. The differentiation of Socs3-deficient progenitor cells is skewed towards macrophage production in response to G-CSF or IL-6, whereas wild-type progenitor cells produce mainly neutrophils. The proliferative capacity of Socs3-deficient progenitor cells is greatly enhanced in response to G-CSF at all concentrations but only at low concentrations for IL-6. Strikingly, synergistic responses to co-stimulation with stem cell factor and IL-6 (but not G-CSF) are lost at higher concentrations in Socs3-deficient progenitor cells. Cytokine-induced expression of transcriptional regulators including cebpb, Ets2, Bcl3, c-Myc, Jun and Fosl2 are differentially regulated in Socs3-deficient cells. The tight regulation by Socs3 of STAT3 phosphorylation and gene transcription after cytokine receptor ligation significantly influences the fate of myeloid progenitor cells.
PMCID: PMC2556307  PMID: 18400361
Suppressor of cytokine signaling-3; cytokines; signal transduction; hematopoiesis; G-CSF

Results 1-25 (614387)