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

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1016-1026 ◽  
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.

scholarly journals Early B Cell Factor Promotes B Lymphopoiesis with Reduced Interleukin 7 Responsiveness in the Absence of E2A

2004 ◽  
Vol 199 (12) ◽  
pp. 1689-1700 ◽  
Author(s):  
Christopher S. Seet ◽  
Rachel L. Brumbaugh ◽  
Barbara L. Kee
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
B Lymphopoiesis ◽  
Protein Activity ◽  
E Proteins ◽  
Helix Loop Helix ◽  
Interleukin 7 ◽  
E Protein ◽  
B Lineage ◽  
Early B Cell Factor

The basic helix-loop-helix transcription factors encoded by the E2A gene function at the apex of a transcriptional hierarchy involving E2A, early B cell factor (EBF), and Pax5, which is essential for B lymphopoiesis. In committed B lineage progenitors, E2A proteins have also been shown to regulate many lineage-associated genes. Herein, we demonstrate that the block in B lymphopoiesis imposed by the absence of E2A can be overcome by expression of EBF, but not Pax5, indicating that EBF is the essential target of E2A required for development of B lineage progenitors. Our data demonstrate that EBF, in synergy with low levels of alternative E2A-related proteins (E proteins), is sufficient to promote expression of most B lineage genes. Remarkably, however, we find that E2A proteins are required for interleukin 7–dependent proliferation due, in part, to a role for E2A in optimal expression of N-myc. Therefore, high levels of E protein activity are essential for the activation of EBF and N-myc, whereas lower levels of E protein activity, in synergy with other B lineage transcription factors, are sufficient for expression of most B lineage genes.

scholarly journals Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2633
Author(s):  
Casper Marsman ◽  
Tineke Jorritsma ◽  
Anja ten Brinke ◽  
S. Marieke van Ham
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Intracellular Signaling ◽  
Simultaneous Detection ◽  
Signaling Proteins ◽  
B Cell Differentiation ◽  
Flow Cytometric ◽  
B Cell Subsets ◽  
Human B Cell

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.

Eto2/MTG16 Regulates E-Protein Activity and Subset Specification in Dendritic Cell Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1229-1229
Author(s):  
Hiyaa Singhee Ghosh ◽  
Kang Liu ◽  
Scott Hiebert ◽  
Boris Reizis
Keyword(s):  
Dendritic Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Dendritic Cell ◽  
Cell Fate ◽  
Cell Lineage ◽  
Transcriptional Regulators ◽  
E Proteins ◽  
E Protein

Abstract Abstract 1229 Eto-family proteins were first discovered as translocation fusion in AML1 (Runx1), a gene most frequently disrupted in human leukemia. Of the translocations that disrupt the AML1 gene in leukemia, Eto1(MTG8)/AML1 translocation accounts for ∼15% of Acute Myeloid Leukemia (AML). The Eto-family proteins function as transcriptional co-repressors that bind to DNA-binding transcription factors to regulate their target genes. Eto2 (MTG16) is an Eto-family member implicated in secondary or therapy-related AML, although recent reports provide evidence for Eto2/MTG16 translocations in de novo AML as well. Furthermore, recent studies have highlighted a role for MTG16 in HSC self renewal and T cell lineage specification, indicating its emerging role overall in hematopoiesis. The co-repressor function of Eto for E-proteins has been described previously in the context of Eto/AML1 fusion proteins. E-proteins are a class of basic-helix-loop-helix (bHLH) transcription factors that play an important role in hematopoiesis. Among the E-protein family, the role of E2A has been extensively studied in B and T cell development. Recently, our lab discovered the specific requirement of the E-protein E2-2 in the development of Plasmacytoid Dendritic Cells (pDC). pDC are the professional interferon producing (IPC) cells of our immune system important in anti-viral, anti-tumor and auto-immunity. pDC are a subtype of the antigen-presenting classical Dendritic Cells (cDC) with distinct structural and functional properties. Recently, we demonstrated that the putative cell fate plasticity of pDC was a direct manifestation of continuous E2-2 function. Using pDC-reporter mice in which E2-2 could be inducibly deleted from mature pDC we showed that the continuous expression of E2-2 was required to prevent the conversion of pDC to cDC. Here we report our current studies that investigate the molecular players underlying the E2-2 orchestrated genetic program for pDC cell fate decision and maintenance. Analyzing the transcriptome of the transitioning pDC, we have identified MTG16 as an important player in the fine regulation of DC lineage decisions. Using knock-out and chimeric mice, progenitor studies, promoter and biochemical analyses, we demonstrate MTG16 as an important E2-2 corepressor, promoting E2-2 mediated genetic program. We report that in order to facilitate the pDC cell fate, MTG16 enables E2-2 to suppress the cDC gene expression program, by negatively regulating the E-protein inhibitor Id2. The cell-fate conversion through deletion or overexpression of lineage-deciding transcriptional regulators has been described previously for B- and T cells. Theseh studies highlight the susceptibility of blood cells to aberrant functions of crucial transcriptional regulators, potentially leading to pathologic conditions. Therefore, understanding the interrelationship between the various genetic regulators that control lineage decisions and cell-fate plasticity is cardinal to accurate diagnosis and therapy for hematopoietic pathologies. Our study provides the first evidence for a physiological role of E-protein/Eto-protein interaction in dendritic cell lineage decision. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

2020 ◽  
Author(s):  
Casper Marsman ◽  
Tineke Jorritsma ◽  
Anja ten Brinke ◽  
Marieke van Ham
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Intracellular Signaling ◽  
Simultaneous Detection ◽  
Signaling Proteins ◽  
B Cell Differentiation ◽  
Flow Cytometric ◽  
B Cell Subsets ◽  
Human B Cell

Flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help elucidate the regulation of B cell survival, proliferation and differentiation. However, simultaneous detection of signaling proteins or TFs, with membrane markers (MM) can be challenging as required fixation and permeabilization procedures can affect functionality of conjugated antibodies. Here, a phosphoflow method is presented for detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6 together with B cell differentiation MM CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows detection of B cell TFs; PAX5, c-MYC, BCL6, AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s together with MM. Applying these methods on in vitro induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and UMAP analysis revealed heterogeneity in TF-expression within stimulated CD27 or CD38-expressing B cell subsets. The methods presented here allow for sensitive analysis of STAT and NF-κB p65 signaling and TFs together with B cell differentiation MM at single-cell resolution. This will aid further investigation of B cell responses in both health and disease.

Involvement of Ets Factor Spi-B, E Protein E2-2, and Id2 in Waldenström's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2946-2946
Author(s):  
Yangsheng Zhou ◽  
Xia Liu ◽  
Lian Xu ◽  
Zachary Hunter ◽  
Jenny Sun ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
B Cell Differentiation ◽  
Protein Activity ◽  
E Proteins ◽  
E Protein ◽  
Nuclear Extracts

Abstract Abstract 2946 Poster Board II-922 Waldenström's macroglobulinemia (WM) is an incurable B cell disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. In this study, we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of BM CD19+ B cells obtained from 13 WM patients with 6 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between the two groups. Transduction of Spi-B in BCWM.1 WM cells resulted in two-fold higher levels of Id2 and five-fold lower levels of E2-2 compared with control. Id2 transduced BCWM.1 cells expressed two-fold lower levels of E2-2 and Spi-B. Taken together, these results implicate that increased expression of Spi-B alone cannot suppress Id2 transcription in the absence of E2-2 activity. Interestingly, overexpressing Spi-B while concomitantly knocking down Id2 increased the expression of the XBP-1 splicing isoform 2.5-fold without changing levels of Blimp-1 and IRF4. Moreover, inhibition of Spi-B expression by RNA interference or forced expression of Id2 in transduced BCWM.1 cells induced a significant decrease of anti-apoptotic Bcl-2. Importantly, we also showed that Spi-B co-immunoprecipated with Blimp-1 in nuclear extracts. Collectively, these data suggest that the regulatory network of the Spi-B, E2-2, and Id2 plays an essential role in B cell differentiation as well as the pathogenesis of WM, and suggests that Spi-B overexpression may block WM cell differentiation by sequestration of Blimp-1 while promoting tumor cell survival though up-regulation of Bcl-2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interleukin-7 is necessary to maintain the B cell potential in common lymphoid progenitors

2005 ◽  
Vol 201 (6) ◽  
pp. 971-979 ◽  
Author(s):  
Sheila Dias ◽  
Hamilton Silva ◽  
Ana Cumano ◽  
Paulo Vieira
Keyword(s):  
B Cell ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Cell Potential ◽  
Differentiation Potential ◽  
Lymphoid Lineage ◽  
Interleukin 7 ◽  
Multipotent Progenitors ◽  
Early B Cell Factor ◽  
Deficient Mice

Interleukin-7 (IL-7) promotes survival and expansion of lymphoid precursors. We show here that, in addition, IL-7 has a fundamental role, as early as the stage of the multipotent (B/T/NK) common lymphoid progenitor (CLP), in maintaining the B cell differentiation program open. CLPs generated in the absence of IL-7 have normal T/NK differentiation potential, but severely impaired B potential. Accordingly, CLPs from IL-7–deficient mice express lower amounts of early B cell factor (EBF) and Pax5 than wild-type CLPs, but similar amounts of GATA-3. Importantly, induced overexpression of EBF is sufficient to restore the B potential in these cells. These results indicate that IL-7 directs commitment of CLPs by modulating EBF expression. This is the first example of a cytokine influencing lymphoid lineage commitment in multipotent progenitors and highlights the relevance of the expression of a functional IL-7 receptor at the CLP stage.

scholarly journals E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Jason H. Rogers ◽  
Kristin S. Owens ◽  
Jeffrey Kurkewich ◽  
Nathan Klopfenstein ◽  
Sangeeta R. Iyer ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Cell Fate ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
B Cell Differentiation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cell Fate Decisions ◽  
Cell Commitment

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.

The MiR23A MicroRNA Cluster Is a Target of PU.1 and Promotes Development of Myeloid Cells at the Expense of B Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2438-2438
Author(s):  
Kristin S Owens ◽  
Kimi Y Kong ◽  
Jason H Rogers ◽  
Richard E. Dahl
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Cell Fate ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Computer Algorithms ◽  
B Cell Differentiation ◽  
Monocytic Differentiation ◽  
Microrna Cluster ◽  
Myeloid Development

Abstract The transcription factor PU.1 is an important regulator of hematopoiesis, directing cell fate decision in a dosage dependent manner with higher levels driving monocytic differentiation and lower levels directing B-cell differentiation. The mechanism by which PU.1 controls this process is not fully understood. Here we show that PU.1 is involved in regulating the expression of several microRNAs. MicroRNAs regulate gene expression at a post-transcriptional level by binding target mRNA through the 3′ UTR and either repressing translation or causing degradation of the mRNA itself. We used microarray expression profiling to assess a large group of microRNAs in PUER cells, a PU.1 −/− cell line that differentiates into macrophages when PU.1 activity is restored. Several miRNAs showed changes in expression four days after restoration of PU.1 activity. We focused on a cluster of microRNAs that includes miR-23a, miR27a, and miR24-2, which we call the miR23a cluster. All three miRNAs are coded for on a single pri-miRNA transcript. Northern blot analysis verified the findings of the microarray–that expression of this microRNA cluster is enhanced by PU.1. The promoter for the cluster contains several conserved predicted binding sites for PU.1. Chromatin immunoprecipitation and EMSA has confirmed that PU.1 binds to these sites. In addition, reporter assays show that the mir23a promoter can be activated by PU.1. The 23a cluster appears to be a critical target gene for PU1 to promote myeloid development. Using in vitro hematopoietic cultures we have observed that expressing this cluster in hematopoietic progenitors promotes myeloid development over B cell development. In addition, data from bone marrow transplant assays will be presented demonstrating the role of this cluster in directing hematopoiesis in vivo. Targetscan and miRanda computer algorithms predict several B cell transcription factors as targets of the miRNAs of the 23a cluster. PU.1 potentially activates expression of the 23a cluster to downregulate B cell transcription factors in order to commit cells to the myeloid cell fate.

scholarly journals Restriction of memory B cell differentiation at the germinal center B cell positive selection stage

2020 ◽  
Vol 217 (7) ◽  
Author(s):  
Amparo Toboso-Navasa ◽  
Arief Gunawan ◽  
Giulia Morlino ◽  
Rinako Nakagawa ◽  
Andrea Taddei ◽  
...  
Keyword(s):  
B Cells ◽  
Positive Selection ◽  
B Cell ◽  
Cell Fate ◽  
Germinal Center ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
B Cell Differentiation ◽  
Repressor Complex ◽  
Selection Stage

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC–MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC–MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC–MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

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