Deletion of genes encoding PU.1 and Spi-B in B cells impairs differentiation and induces pre-B cell acute lymphoblastic leukemia

Abstract

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, ...

Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors

“…4B). SPIB and SPI1 have the same canonical motif (PU.1) and are both essential for B cell development (Sokalski et al 2011). (2) GATA1 was the most frequently discovered noncanonical motif in K562 cells.…”

Regulation of B Cell Linker Protein Transcription by PU.1 and Spi-B in Murine B Cell Acute Lymphoblastic Leukemia

“…We previously noted that levels of Blnk mRNA transcripts were reduced in sorted splenic DPB B-ALL cells compared with control B cells (15). Therefore, we hypothesized that Blnk is a target of transcriptional activation by PU.1 and/or Spi-B.…”

“…Reduced Spi-B expression was recently associated with human B-ALL (14). Our laboratory previously showed that mice that lack both PU.1 and Spi-B in the B cell lineage (CD19 +/Cre Sfpi1 lox/lox Spib 2/2 mice, henceforth known as DPB mice) have impaired B cell development and develop B-ALL with 100% incidence rate by 21 wk of age (15). These results demonstrate that PU.1 and Spi-B have tumorsuppressor function in the B cell lineage and suggest that mutation of the SPI1 and SPIB genes, or upstream activators of these genes, could be oncogenic drivers in leukemia-initiating cells.…”

The RUNX1–PU.1 axis in the control of hematopoiesis

“…On the other hand, RUNX1 is downregulated or mutated in TLX1 T-ALL, as its normal expression entails tumorsuppressing effects [92]. Conversely, the role of PU.1 in ALL is less established, although it is expressed in most B-ALLs, and that its deletion together with SpiB systematically results in leukemia [93,94]. In T-ALL recent studies have reported expression of PU.1; however, the mechanisms in which it could contribute towards leukemogenesis remain unclear [93,95].…”