Abstract
Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1+/− Spib−/− [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00117-15.
ACKNOWLEDGMENTS
This work was supported by grants from the Canadian Institutes of Health Research (MOP-106581) and the National Sciences and Engineering Research Council (grant 386046) to R.P.D. and by an OGSST scholarship to S.K.H.L.
We thank Edwin Lee-Chan and Enayat Nikoopour for assistance in performing tritiated thymidine incorporation assays. We acknowledge assistance of the London Regional Genomics core facility with DNA sequencing and Kristin Chadwick from London Regional Flow Cytometry for performing cell sorting. We acknowledge the University of British Columbia Cancer Research Agency and Genome Quebec for assistance with ChIP-seq. We thank Jan Piskorz for reanalysis of ChIP-seq data and Steven Kerfoot for critically reading the manuscript. We acknowledge Stephen Smale for the contribution of the p50 cFlag pcDNA3 and MIG-cRel vectors.