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

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.

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

Cells ◽

10.3390/cells9122633 ◽

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.

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

10.20944/preprints202012.0028.v1 ◽

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.

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Fra-2 regulates B cell development by enhancing IRF4 and Foxo1 transcription

Journal of Experimental Medicine ◽

10.1084/jem.20160514 ◽

2017 ◽

Vol 214 (7) ◽

pp. 2059-2071 ◽

Cited By ~ 11

Author(s):

Kenia Ubieta ◽

Mireia Garcia ◽

Bettina Grötsch ◽

Steffen Uebe ◽

Georg F. Weber ◽

...

Keyword(s):

Cell Proliferation ◽

Transcription Factors ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Gene Profiling ◽

B Cell Differentiation

The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7–stimulated pro–B cell cultures revealed a reduced differentiation from large pre–B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2–deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro–B cell proliferation and small pre–B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2–deficient pro–B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

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