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 acute lymphoblastic leukemia (B-ALL) is frequently associated with mutations or chromosomal translocations of genes encoding transcription factors. Conditional deletion of genes encoding the E26-transformation–specific transcription factors, PU.1 and Spi-B, in B cells (ΔPB mice) leads to B-ALL in mice at 100% incidence rate and with a median survival of 21 wk. We hypothesized that PU.1 and Spi-B may redundantly activate transcription of genes encoding tumor suppressors in the B cell lineage. Characterization of aging ΔPB mice showed that leukemia cells expressing IL-7R were found in enlarged thymuses. IL-7R–expressing B-ALL cells grew in culture in response to IL-7 and could be maintained as cell lines. Cultured ΔPB cells expressed reduced levels of B cell linker protein (BLNK), a known tumor suppressor gene, compared with controls. The Blnk promoter contained a predicted PU.1 and/or Spi-B binding site that was required for promoter activity and occupied by PU.1 and/or Spi-B as determined by chromatin immunoprecipitation. Restoration of BLNK expression in cultured ΔPB cells opposed IL-7–dependent proliferation and induced early apoptosis. We conclude that the tumor suppressor BLNK is a target of transcriptional activation by PU.1 and Spi-B in the B cell lineage.