E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of theMCSFRpromoter through antagonizing PU.1’s ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

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Balance between Id and E proteins regulates myeloid-versus-lymphoid lineage decisions

Blood ◽

10.1182/blood-2008-06-164996 ◽

2009 ◽

Vol 113 (5) ◽

pp. 1016-1026 ◽

Cited By ~ 42

Author(s):

Shawn W. Cochrane ◽

Ying Zhao ◽

Robert S. Welner ◽

Xiao-Hong Sun

Keyword(s):

Transcription Factors ◽

B Cell ◽

Cell Fate ◽

Intracellular Signaling ◽

Specific Gene ◽

B Cell Differentiation ◽

E Proteins ◽

Lymphoid Lineage ◽

E Protein ◽

Multipotent Progenitors

Abstract Hematopoiesis consists of a series of lineage decisions controlled by specific gene expression that is regulated by transcription factors and intracellular signaling events in response to environmental cues. Here, we demonstrate that the balance between E-protein transcription factors and their inhibitors, Id proteins, is important for the myeloid-versus-lymphoid fate choice. Using Id1-GFP knockin mice, we show that transcription of the Id1 gene begins to be up-regulated at the granulocyte-macrophage progenitor stage and continues throughout myelopoiesis. Id1 expression is also stimulated by cytokines favoring myeloid differentiation. Forced expression of Id1 in multipotent progenitors promotes myeloid development and suppresses B-cell formation. Conversely, enhancing E-protein activity by expressing a variant of E47 resistant to Id-mediated inhibition prevents the myeloid cell fate while driving B-cell differentiation from lymphoid-primed multipotent progenitors. Together, these results suggest a crucial function for E proteins in the myeloid-versus-lymphoid lineage decision.

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Logical modeling of lymphoid and myeloid cell specification and transdifferentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1610622114 ◽

2017 ◽

Vol 114 (23) ◽

pp. 5792-5799 ◽

Cited By ~ 51

Author(s):

Samuel Collombet ◽

Chris van Oevelen ◽

Jose Luis Sardina Ortega ◽

Wassim Abou-Jaoudé ◽

Bruno Di Stefano ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Regulatory Network ◽

Developmental Process ◽

Ectopic Expression ◽

Myeloid Cell ◽

Hematopoietic Stem ◽

Chip Sequencing ◽

Cell Fate Decisions ◽

Public Data

Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.

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E47 Binds to PU.1 Inhibiting Its Ability To Bind DNA and Activate Gene Expression.

Blood ◽

10.1182/blood.v104.11.1609.1609 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1609-1609

Author(s):

Richard Dahl ◽

Sangeeta R. Iyer ◽

M. Celeste Simon

Keyword(s):

B Cell ◽

Cell Fate ◽

Cell Lineage ◽

Myeloid Cell ◽

Receptor Protein ◽

Cell Fate Decisions ◽

Myeloid Development ◽

Specific Factors ◽

Low Levels ◽

Activate Gene

Abstract The transciption factor PU.1 is required for the development of macrophages, granulocytes, and B lymphocytes. Additionally its protein concentration in multipotential hematopoietic progenitor cells regulates cell fate decisions with high levels of PU.1 directing myeloid cell fate acquisition and low levels directing B cell fate acquisition. Potentially high levels of PU.1 are required for myeloid development in order to overcome repressive effects of B cell lineage specific factors. The essential B cell factor BSAP (Pax-5) was shown to associate with PU.1 and repress its transactivation activity. Here we investigate whether two other essential B cell factors, E2A and EBF could affect PU.1 activity. We observed that the E2A component, E47, but not EBF could associate with PU.1. Additionally E47 could repress PU.1 dependent transactivation of the M-CSFR promoter and that this repression is mediated by inhibiting PU.1 DNA binding. Exogenous E47 expression in an IL-3 dependent multipotential cell line could block PU.1 induced differentiation and expression of endogenous M-CSF receptor protein. Our data suggests that high PU.1 concentration is required for myeloid development in order to overcome repressive actions of other lineage specific transcriptional regulators such as E47.

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Regulation and Function of NOTCH2 in B-CLL Cells.

Blood ◽

10.1182/blood.v110.11.1138.1138 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1138-1138

Author(s):

Rainer Hubmann ◽

Martin Hilgarth ◽

Susanne Schnabl ◽

Dita Demirtas ◽

Josef D. Schwarzmeier ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Fate ◽

Target Gene ◽

Active Form ◽

Lymphocytic Leukemia ◽

B Cell Differentiation ◽

Cell Fate Decisions ◽

Mutational Status ◽

And Function

Abstract B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI’s) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

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Temporal and Sequential Expression of EBF1 and PAX5 Restricts the Non-B Cell Fate In Early Lymphopoiesis

Blood ◽

10.1182/blood.v116.21.3867.3867 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3867-3867

Author(s):

Sasan Zandi ◽

Jenny Zetterblad ◽

Panagiotis Tsapogas ◽

Robert Månsson ◽

Mikael Sigvardsson

Keyword(s):

Transcription Factors ◽

T Cell ◽

B Cell ◽

Cell Fate ◽

B Cell Lymphoma ◽

Cell Development ◽

B Cell Development ◽

Cell Potential ◽

Lymphoid Development ◽

Cell Commitment

Abstract Abstract 3867 The generation of B cells from multipotent hematopoietic stem cells involves activation of B cell specific and repression of alternative lineage programs. This process to a large extent is under the control of transcription factors such as EBF1, E2A, IKAROS and PAX5. Inactivation of PAX5 and EBF1, by point mutations or deletions can be found in 30% of the acute lymphocytic leukemia (ALL) cases, probably due to a disturbance of differentiation in early B cell development. Conditional deletion of PAX5 and EBF1 leads to a complete block in B cell production and subsequent accumulation of early immature B cell progenitors. Although the roles of these transcription factors have been established in B cell development, the presence of a feedback loop between PAX5 and EBF1 as well as lack of well defined early B cell developmental events and cell populations, lead to some discrepancies in the current literature regarding the role of these factors in restricting non-B cell lineages and the establishment of B cell fate. By using a combination of RAG1/EBF1 reporter mice and newly identified surface markers i.e Ly6D, we have shown the exact point of myeloid (M), natural killer (NK) and T cells restriction within conventional common lymphoid progenitors (CLP) population. The Lin-IL7r+flt3+kitloSca1lo(CLP)Ly6D-λ5- population retains the residue of myeloid potential together with dendritic (D), Nk, T and B potentials. Upon expression of Ly6D, these cells lose the residual myeloid, NK and D potentials, and eventually the expression of λ5, associated with loss of T cell potential, marks the B-cell committed cells. Based on this new model of lymphoid development we have revisited the functional roles of EBF1 and PAX5 in B-cell commitment. This work suggests that EBF1 restricts the myeloid, NK and D potentials in the transition from CLPLy6D- to CLPLy6D+ stage while the expression of PAX5 in the transition from CLPLy6D+λ5- to CLPLy6D+λ5+ restricts the T cell potential by possibly by counteracting Notch1 signaling in the bone marrow. Considering the high prevalence of PAX5 and EBF1 mutations in ALL and diffuse large B cell lymphoma (DLBL) cases and the fact that inactivation of PAX5 and EBF1 leads to accumulation of B cell progenitors in mice models, understanding the temporal and spatial expression of EBF1 and PAX5 in regard to restriction of non-B cell fates in early stages of lymphoid development will allow us to identify the leukemia-initiating cells and the mechanisms of leukemogenesis in these diseases. Disclosures: No relevant conflicts of interest to declare.

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Autoreactive T cells preferentially drive differentiation of non-responsive memory B cells at the expense of germinal center maintenance

10.1101/287789 ◽

2018 ◽

Author(s):

Rajiv W Jain ◽

Kate A Parham ◽

Yodit Tesfagiorgis ◽

Heather C Craig ◽

Emiliano Romanchik ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Germinal Center ◽

Memory B Cells ◽

B Cell Differentiation ◽

Cell Fate Decisions ◽

The Status

AbstractB cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG), and compare them the response to a standard model foreign antigen (NP-haptenated ovalbumin, NPOVA). Both antigens generated productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response the status of the cognate T cell partner drove preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation was largely independent of T cell influence. Interestingly, memory B cells formed in the MOG GC were unresponsive to secondary challenge and this could not be overcome with T cell help.

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A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.

Blood ◽

10.1182/blood.v108.11.1152.1152 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1152-1152

Author(s):

Rita Fragoso ◽

Catia Igreja ◽

Claudia Appleton ◽

Alexandra Henriques ◽

Nuno Clode ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Myeloid Differentiation ◽

B Cell Differentiation ◽

Cd34 Cells ◽

Cell Commitment

Abstract VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p<0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid differentiation.

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Cell Fate Decisions: The Role of Transcription Factors in Early B-cell Development and Leukemia

Blood Cancer Discovery ◽

10.1158/2643-3230.bcd-20-0011 ◽

2020 ◽

Vol 1 (3) ◽

pp. 224-233

Author(s):

Ute Fischer ◽

Jun J. Yang ◽

Tomokatsu Ikawa ◽

Daniel Hein ◽

Carolina Vicente-Dueñas ◽

...

Keyword(s):

Transcription Factors ◽

B Cell ◽

Cell Fate ◽

Cell Development ◽

B Cell Development ◽

Cell Fate Decisions

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The MADS transcription factor Mef2c is a pivotal modulator of myeloid cell fate

Blood ◽

10.1182/blood-2007-10-116343 ◽

2008 ◽

Vol 111 (9) ◽

pp. 4532-4541 ◽

Cited By ~ 37

Author(s):

Andrea Schüler ◽

Maike Schwieger ◽

Afra Engelmann ◽

Kristoffer Weber ◽

Stefan Horn ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Cell Fate ◽

Serum Response Factor ◽

Myeloid Cell ◽

Cardiovascular Development ◽

Monocytic Differentiation ◽

Cell Fate Decisions ◽

Extracellular Stimuli ◽

Interfering Rna

AbstractMef2c is a MADS (MCM1-agamous–deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML, this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis, whereas BM isolated from Mef2cΔ/− mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun, but not PU.1, C/EBPα, or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover, retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation, coupled with its functional sensitivity to extracellular stimuli, demonstrate an important role in immunity—and, consistent with findings of other myeloid transcription factors, a target of oncogenic lesions in AML.

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Receptor Interacting Protein Kinase Pathways Regulate Innate B Cell Developmental Checkpoints But Not Effector Function in Mice

Frontiers in Immunology ◽

10.3389/fimmu.2021.758407 ◽

2021 ◽

Vol 12 ◽

Author(s):

Raksha Parthasarathy ◽

Thomas Hägglöf ◽

Jason T. Hadley ◽

Alexandra McLennan ◽

Aiden Mattke ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Fate ◽

Cell Types ◽

Effector Function ◽

Caspase 8 ◽

B Cell Differentiation ◽

Autoantibody Production ◽

Interacting Protein ◽

Cell Commitment

Mutations in the scaffolding domain of Receptor Interacting Protein kinases (RIP) underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIP1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIP1, RIP3, and caspase-8 triple deficient and RIP3, caspase-8 double deficient mice, we identified selective contributions of RIP1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIP1, RIP3, and caspase-8 sufficiency. RIP1 regulated MZ B cell development rather than differentiation and RIP1 mediates its innate immune effects independent of the RIP1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of mice doubly deficient in both caspase-8 and RIP3 or deficient in all three proteins (RIP3, caspase-8, and RIP1) revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to WT mice. Thus, RIP kinases and caspase-8 jointly orchestrate B cell fate and delayed effector function through a B cell-intrinsic mechanism.

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