The E26 transformation-specific (Ets) transcription factor PU.1 is required to generate lymphoid progenitor cells from hematopoietic stem cells, but it is not required to generate B cells from committed B-cell lineage progenitors. We hypothesized that PU.1 function in B-cell differentiation is complemented by the related Ets transcription factor Spi-B. To test this hypothesis, mice were generated lacking both PU. IntroductionThe E26 transformation-specific (Ets) transcription factor PU.1 (encoded by the gene Sfpi1 in mice and SPI1 in humans) is required for generating lymphoid progenitor cells and is a key regulator of B-cell fate specification. 1,2 However, conditional deletion of the Sfpi1 gene under control of the B cell-specific Cd19 locus results in minimal perturbation of B-cell development and function. 3,4 Spi-B (encoded by Spib) is expressed in developing B cells 5 and interacts with DNA binding sites identical to PU.1. 6 Several studies suggest that PU.1 and Spi-B functions are complementary. Spi-B can partially replace PU.1 in myeloid and B-cell differentiation. 7,8 In addition, Sfpi1 ϩ/Ϫ Spib Ϫ/Ϫ mice have severely reduced frequencies of splenic follicular (FO) B cells compared with either Sfpi1 ϩ/Ϫ or Spib Ϫ/Ϫ mice. 9 We hypothesized that PU.1 is partially redundant after B-cell commitment because of complementation by Spi-B. To test this hypothesis, we generated mice that delete a conditional Sfpi1 lox allele under control of the B cell-specific Cd19 locus on a Spib Ϫ/Ϫ background (CD19 ϩ/Cre Sfpi1 lox/lox Spib Ϫ/Ϫ mice). We report here that CD19 ϩ/Cre Sfpi1 lox/lox Spib Ϫ/Ϫ mice have reduced frequencies of B cells and impaired B-cell differentiation. Strikingly, all CD19 ϩ/Cre Sfpi1 lox/lox Spib Ϫ/Ϫ mice develop pre-B cell acute lymphoblastic leukemia (ALL) with thymic involvement before 30 weeks of age. These findings demonstrate that PU.1 and Spi-B have complementary function as essential transcriptional regulators and novel tumor suppressors in the B-cell lineage. MethodsBreeding and care of mice CD19 Cre/Cre mice 10 were purchased from The Jackson Laboratory (stock no. 006785) and mated to Sfpi1 lox/lox mice 2 to generate CD19 ϩ/Cre Sfpi1 ϩ/lox mice.These were mated to Spib Ϫ/Ϫ mice 11 to generate CD19 ϩ/Cre Sfpi1 ϩ/lox Spib ϩ/Ϫ mice that were intercrossed to generate CD19 ϩ/Cre Sfpi1 lox/lox Spib Ϫ/Ϫ mice. Mice used in this study were on the C57Bl/6 background and were generated by mating CD19 ϩ/Cre Sfpi1 lox/lox males to CD19 ϩ/ϩ Sfpi1 lox/lox females or by mating CD19 ϩ/Cre Sfpi1 lox/lox Spib Ϫ/Ϫ males to CD19 ϩ/ϩ Sfpi1 lox/lox Spib Ϫ/Ϫ females. Mouse care was monitored under an approved animal use subcommittee protocol in accord with the University of Western Ontario Council on Animal Care. PCR and genotypingGenotyping of mice was performed using standard PCR. Quantitative PCR (qPCR) was performed using a Rotor-Gene 6000 instrument (Corbett Life Sciences). The relative frequency of intact Sfpi1 lox or deleted Sfpi1⌬ alleles was normalized to the frequency of -actin promoter genomic DNA, ...
B cell development and Ig rearrangement are governed by cell type- and developmental stage-specific transcription factors. PU.1 and Spi-B are E26-transformation-specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)-deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B-deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation-sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igκ locus, including Vκ promoters and regions located downstream of Vκ second exons. Induction of PU.1 induced Igκ transcription and rearrangement. Upregulation of Igκ transcription was impaired in small pre-B cells from PU.1/Spi-B-deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igκ transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development.
BackgroundSpi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, roles in B cell development. PU.1 and Spi-B are both expressed in B cells, and have been demonstrated to redundantly activate transcription of genes required for B cell differentiation and function. It was hypothesized that Spi-B and PU.1 occupy a similar set of regions within the genome of a B lymphoma cell line.ResultsTo compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. The majority of regions occupied by Spi-B were also occupied by PU.1. Regions bound by Spi-B and PU.1 were frequently located immediately upstream of genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif.ConclusionsOur results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1303-0) contains supplementary material, which is available to authorized users.
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