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A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation

Abstract

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.

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Figure 1: The IL-7R–PI(3)K–Akt pathway negatively regulates Foxo activity and the expression of Rag1 and Rag2 in pre-B cells.
Figure 2: ChIP-seq analysis of the Foxo1 cistrome in Irf4−/−Irf8−/− pre-B cells.
Figure 3: Foxo1 is necessary for the induction of two key components of pre-BCR signaling.
Figure 4: Foxo1 and Pax5 act together to induce Blnk expression after attenuation of IL-7 signaling.
Figure 5: A positive feedback loop of Foxo1-BLNK-p38 regulates the differentiation of pre-B cells.
Figure 6: Activation of the Foxo1-BLNK-p38 module is specific to pre-B cells.
Figure 7: A gene-regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation.

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Acknowledgements

We thank S. Heinz for advice on the preparation of DNA libraries for ChIP-seq; M. Veselitis (University of Chicago) for mouse Blnk cDNA; S.E. Powers for discussions; the Bendelac laboratory for assistance with flow cytometry; R. Duggan, D. Leclerc, M. Olson and J. Cao for assistance with cell sorting; and K. Michelini and J. Zekos for operating the Illumina Genome Analyzer II at the University of Chicago (supported by the Howard Hughes Medical Institute). Supported by Howard Hughes Medical Institute (H.S.), the US National Institutes of Health (GM088847 to M.R.C. and GM081892 to A.R.D.) and the US Department of Energy (DOE) Computational Science Graduate Fellowship Program (M.M.-C.).

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Authors

Contributions

K.O. and H.S. designed the experiments; K.O. did most of the experiments; M.M.-C. created the program for ChIP-seq data analysis; M.M. analyzed the abundance of phosphorylated Akt in pre-B cells and in Rag2−/− pro-B cells reconstituted with μH383; J.R.T. assisted with ChIP-seq data analysis; E.B. assisted with electrophoretic mobility-shift assays; M.R.C., A.R.D. and R.S. provided experimental advice and input on the manuscript; and K.O. and H.S. wrote the manuscript.

Corresponding author

Correspondence to Harinder Singh.

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H.S. is an employee of Genentech.

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Ochiai, K., Maienschein-Cline, M., Mandal, M. et al. A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation. Nat Immunol 13, 300–307 (2012). https://doi.org/10.1038/ni.2210

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