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 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 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.

scholarly journals Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

1991 ◽  
Vol 11 (10) ◽  
pp. 4885-4894 ◽  
Author(s):  
C L Miller ◽  
A L Feldhaus ◽  
J W Rooney ◽  
L D Rhodes ◽  
C H Sibley ◽  
...  
Keyword(s):  
B Cell ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
De Novo ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Immunoglobulin Gene ◽  
B Cell Differentiation ◽  
Nf Kappa B ◽  
Specific Expression

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

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.

scholarly journals IFI16Expression Is Related to Selected Transcription Factors during B-Cell Differentiation

2015 ◽  
Vol 2015 ◽  
pp. 1-20 ◽  
Author(s):  
Pier Paolo Piccaluga ◽  
Claudio Agostinelli ◽  
Fabio Fuligni ◽  
Simona Righi ◽  
Claudio Tripodo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Peripheral Blood Lymphocytes ◽  
B Cell Differentiation ◽  
Cellular Survival ◽  
Human B Cells ◽  
Proliferation And Differentiation ◽  
B Cell Subsets

The interferon-inducible DNA sensor IFI16 is involved in the modulation of cellular survival, proliferation, and differentiation. In the hematopoietic system, IFI16 is consistently expressed in the CD34+ stem cells and in peripheral blood lymphocytes; however, little is known regarding its regulation during maturation of B- and T-cells. We explored the role of IFI16 in normal B-cell subsets by analysing its expression and relationship with the major transcription factors involved in germinal center (GC) development and plasma-cell (PC) maturation.IFI16mRNA was differentially expressed in B-cell subsets with significant decrease inIFI16mRNA in GC and PCs with respect to naïve and memory subsets.IFI16mRNA expression is inversely correlated with a few master regulators of B-cell differentiation such asBCL6, XBP1, POU2AF1, andBLIMP1. In contrast,IFI16expression positively correlated withSTAT3, REL, SPIB, RELA, RELB, IRF4, STAT5B, andSTAT5A. ARACNE algorithm indicated a direct regulation ofIFI16byBCL6,STAT5B, andRELB, whereas the relationship betweenIFI16and the other factors is modulated by intermediate factors. In addition, analysis of the CD40 signaling pathway showed thatIFI16gene expression directly correlated with NF-κB activation, indicating that IFI16 could be considered an upstream modulator of NF-κB in human B-cells.

MAZ Is a Substrate for the Deubiquitinating Enzyme DUB1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3913-3913
Author(s):  
Yong-Soo Kim ◽  
Dong-Mi Shin ◽  
Hongsheng Wang ◽  
Herbert Morse
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Deubiquitinating Enzymes ◽  
Transfected Cells

Abstract Abstract 3913 Ubiquitination is a fundamental mechanism of signal transduction that regulates immune responses and many other biological processes. Similar to phosphorylation, ubiquitination is a reversible process that is counter-regulated by ubiquitinating enzymes and deubiquitinating enzymes (DUBs). DUB1 has been identified as a member of a subfamily of deubiquitinating enzymes that are induced by Interleukin-3 (IL-3) in Ba/F3 cells. Recently, we've known that DUB1 is expressed in some pro-B and pre-B cell lines and is differentially regulated during normal B cell differentiation with highest expression in small pre-B cells. To understand the functional role of DUB1 in early B cell development, we identified a transcription factor, MAZ, as an interacting partner or substrate of DUB1 in the Abelson-transformed 220-8 pro-B cell line by using a retrovirus-based protein-fragment complementation assay (RePCA) screen. MAZ has been identified as a critical regulator of p21 gene induction, which controls cell cycle progression in synovial fibroblast cells. Immunoprecipitation and immunoblot analysis confirmed that MAZ and DUB1 interact with each other in cells. DUB1 expression significantly increased the steady-state cellular levels of MAZ. Notably, the half-life of MAZ in the pEGFP-DUB1-transfected cells was significantly increased by DUB1 expression, whereas the half-life of MAZ in the mock-transfected cells was less than 1 hr. These data therefore demonstrate that DUB1 specifically stabilizes MAZ in vivo. We found that the overexpressed MAZ was polyubiquitinated and that the polyubiquitinated MAZ was the substrate for proteasome inhibitor MG132 caused a robust increase of MAZ polyubiquitination. Overexpression of DUB1 in 293T cells caused a decrease of MAZ polyubiquitination in a DUB1 dose-dependent manner. Taken together, these results indicate that DUB1 mediate the ubiquitination-dependent degradation of MAZ. Other study shows that the DUB1 targets both K-48 and K-63 linked ubiquitination suggesting that it may be involved in both protein degradative and non-degradative functions during early B cell differentiation. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of MEF2C-Dependent Transcription in Lymphoid Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4341-4341
Author(s):  
Nikki R. Kong ◽  
Li Chai ◽  
Astar Winoto ◽  
Robert Tjian
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Progenitor Cells ◽  
Luciferase Reporter ◽  
B Cell Differentiation ◽  
Reporter Assays ◽  
B Lineage ◽  
Transcription Program

Abstract Hematopoiesis is a multi-step developmental process that requires an intricate coordination of signal relays and transcriptional regulation to give rise to all blood lineages in the organism. Hematopoietic stem/progenitor cells (HSPCs) can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for HSPCs is whether to adopt the lymphoid or myeloid fate. Despite the discovery of several transcription factors required for different lineages of hematopoietic differentiation, the understanding of how gene expression allows HSPCs to adopt the lymphoid fate still remains incomplete. A study using an inducible hematopoietic-specific (Mx1-Cre) KO mouse line found that Myocyte Enhancer Factor 2C (MEF2C) is required for multi-potent HSPCs to differentiate into the lymphoid lineage (Stehling-Sun et al, 2009). However, the mechanisms of how MEF2C is activated and in turn, drives lymphoid fate specification are not known. Through a candidates approach with co-expression and co-immunoprecipitation, we have identified Early B Cell Factor 1 (EBF1) to be a specific interacting partner of MEF2C, and not other B cell specific transcription factors such as E12, E47, or PAX5. Genome-wide survey of MEF2C and EBF1 binding sites via chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in a proB cell line revealed that these two sequence-specific transcription factors co-occupy the promoters and intragenic regions of many B cell specific genes such as Il7ra, Myb, Foxo1, Ets1, Ebf1 itself, and Pou2af1. Regulatory regions of Il7ra and Foxo1 derived from the ChIP-seq data, as well as an artificial enhancer containing trimerized MEF2C and EBF1 binding sites, were examined in luciferase reporter assays and found to be sufficient to drive transcription from a minimal reporter in 293T cells. Further, this activation was co-dependent on MEF2C and EBF1 expression. The functional relevance of MEF2C binding was further supported by gene expression analyses of MEF2C-regulated B lineage genes in Mx1-Cre Mef2c KO mice compared to WT mice. Consistent with ChIP-seq and luciferase reporter assays, Myb, Ebf1, Il7ra, and Foxo1 all had significantly decreased expression levels in MEF2C-null HSPCs as well as B lineage progenitor cells, compared to sex-matched littermate control mice. Interestingly, myeloid gene expression in Mef2c-KO mice was increased compared to WT control. MEF2C ChIP-seq in a murine HSPC line revealed that it binds myeloid lineage gene targets that are not regulated by MEF2C in proB cells. These results suggest that MEF2C can repress myeloid gene expression in HSPCs. To elucidate the mechanism of this functional switch, we tested the requirement for MAPK pathways to phosphorylate and activate MEF2C at three previously identified residues in order to drive B cell differentiation. Inhibition of p38 MAPK (p38i), but not ERK1/2/5, decreased the potential of HSPCs to differentiate into B220+CD19+ B cells cultured with cytokines that drive this particular lineage fate. Instead, p38i-treated progenitor cells gave rise to more myeloid cells. 65% of this phenotype was rescued by over-expressing a phosphomimetic mutant of MEF2C that can bypass p38 inhibition. Furthermore, MEF2C is known to bind class II HDAC proteins to repress gene expression, providing a possible mechanism for its repression of myeloid transcription program. Supporting this mechanism, phosphomimetic and HDAC-binding double mutant of MEF2C can rescue p38 inhibition phenotype almost 100%. Taken together, this study elucidated the molecular mechanisms of a key lymphoid-specific lineage fate determinant, MEF2C. We discovered that p38 MAPK converts MEF2C from a transcriptional repressor to an activator by phosphorylation in B cell specification, which can be applied to understanding other cell differentiation processes regulated by this important stress-induced signaling pathway. Furthermore, we identified MEF2C’s binding and co-activating partner EBF1, several novel B cell specific targets that it activates in proB cells, and a novel myeloid transcription program that it represses in hematopoietic progenitors. Therefore, these results expanded our understanding of the intricate transcription network that regulates B cell differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intracellular sterol sensing controls intestinal B cell differentiation

2020 ◽  
Author(s):  
Bruno C. Trindade ◽  
Simona Ceglia ◽  
Alyssa Berthelette ◽  
Fiona Raso ◽  
Kelsey Howley ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cells ◽  
Cell Metabolism ◽  
Dietary Cholesterol ◽  
Effector Function ◽  
B Cell Differentiation ◽  
Environmental Signals ◽  
Iga Response

AbstractIntestinal B cell responses are critical for the maintenance of gut homeostasis, yet environmental signals that control B cell metabolism and effector function remain poorly characterized. Here, we show that Peyer’s patches germinal center (GC) B cells are sensitive to 25-hydroxycholesterol (25-HC), an oxidized metabolite of cholesterol produced by the enzyme cholesterol 25-hydroxylase (CH25H). In mice lacking CH25H, antigen-specific GC B cells show an increased cholesterol metabolic signature and preferentially differentiate in plasma cells (PCs), thereby inducing a stronger intestinal IgA response upon immunization or infection. GC B cells express the sterol sensor SREBP2 and use it to sense 25-HC. Deletion of SREBP2 from GC B cells prevents PC differentiation and forces the maintenance of GC identity. GC localized oxysterol production by follicular dendritic cells is central in dictating GC metabolism and imposing B cell fate. Our findings show that the 25-HC-SREBP2 axis shapes B cell effector function in intestinal lymphoid organs and indicate that dietary cholesterol can instruct local B cell response.

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