Objective-It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1) promotes macrophage development, whereas low concentration induces B cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon which resulted in a reduction of PU.1 expression in order to test this hypothesis. Methods-Using gene targeting in ES cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence activated cell sorting, microscopy, and colony forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo.Results-Lymphoid and myeloid cells derived from cultured Sfpi1 BN/BN fetal liver cells had reduced levels of PU.1 expression and activity. B cell development was intrinsically blocked in cells isolated from Sfpi1 BN/BN mice. In addition, myeloid development was impaired in Sfpi1 BN/BN fetal liver. However, neonatal Sfpi1 BN/BN mice had a dramatic expansion and infiltration of immature myeloid cells.Conclusion-Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1−/− fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcγRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcγRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcγRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3′ regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

The Ets family transcription factor PU