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

Abstract

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.

PU.1 Opposes IL-7–Dependent Proliferation of Developing B Cells with Involvement of the Direct Target Gene Bruton Tyrosine Kinase

“…Our laboratory previously showed that Blnk (encoding B cell linker protein) is a direct target of PU.1 and Spi-B in developing B cells (17). Loss-of-function mutation of Btk strongly synergizes with loss-of-function mutation of Blnk in mice to induce B-ALL in mice with ∼75% incidence by 16 wk of age (20).…”

“…Our laboratory showed that combined null mutation of Spi1 and Spib in mice (CD19 +/Cre Spi1 lox/lox Spib 2/2 mice, called CD19-CreDPB mice in this study) leads to B-ALL at a median age of 21 wk at 100% incidence (16). The tumor sup-pressor gene Blnk is a direct target of activation by PU.1 and/or Spi-B (17). However, it is not clear how reduced PU.1 and Spi-B predispose developing B cells to malignant transformation in CD19-CreDPB mice.…”

Nfkb1 Activation by the E26 Transformation-Specific Transcription Factors PU.1 and Spi-B Promotes Toll-Like Receptor-Mediated Splenic B Cell Proliferation

“…Site-directed mutagenesis was performed on the predicted PU.1/Spi-B binding sites of the two closest sites of the TSS of the promoter, by replacing the GGAA (TTCC on complement strand) binding site with GGAC (GTCC) (Fig. 6D), which was previously reported to abolish PU.1 binding (13,20). Luciferase reporter assays showed that the Nfkb1 promoter was active in WEHI-279 cells, and mutation of PU.1/Spi-B sites significantly reduced transcriptional activation (Fig.…”

“…PU.1 and Spi-B share 67% amino acid homology in their DNA binding domain and can bind an identical consensus sequence containing the core motif 5=-GGAA-3= (16-18). Both transcription factors are expressed in B cells, regulate common target genes, and are functionally redundant (19,20 In this study, it was determined whether PU.1 and Spi-B regulate innate immune responses in B cells. Impairment in TLR-mediated proliferation in PUB B cells was observed.…”

“…PU.1 and Spi-B share 67% amino acid homology in their DNA binding domain and can bind an identical consensus sequence containing the core motif 5=-GGAA-3= (16)(17)(18). Both transcription factors are expressed in B cells, regulate common target genes, and are functionally redundant (19,20).…”