The B29 (immunoglobulin beta-chain) gene is a genetic target for early B-cell factor

Abstract

Early B-cell factor (EBF) is a transcription factor suggested as essential for early B-lymphocyte development by findings in mice where the coding gene has been inactivated by homologous disruption. This makes the identification of genetic targets for this transcription factor pertinent for the understanding of early B-cell development. The lack of B29 transcripts, coding for the beta subunit of the B-cell receptor complex, in pro-B cells from EBF-deficient mice suggested that B29 might be a genetic target for EBF. We here present data suggesting that EBF interacts with three independent sites within the mouse B29 promoter. Furthermore, ectopic expression of EBF in HeLa cells activated a B29 promoter-controlled reporter construct 13-fold and induced a low level of expression from the endogenous B29 gene. Finally, mutations in the EBF binding sites diminished B29 promoter activity in pre-B cells while the same mutations did not have as striking an effect on the promoter function in B-cell lines of later differentiation stages. These data suggest that the B29 gene is a genetic target for EBF in early B-cell development.

FIG. 1

The B29 promoter contains three independent EBF binding sites. (A) Schematic drawing of the B29 minimal promoter as defined by Omori and Wall (25). The potential EBF binding sites are underlined, and a consensus EBF binding site (34) is shown. (B) EMSA in which the binding of in vitro-translated recombinant EBF to the mb-1 promoter binding sites was competed for by potential EBF binding sites from the B29 promoter. (C) EMSA in which the binding of in vitro-translated recombinant EBF to the mb-1 promoter binding site was competed for by EBF binding sites from the mb-1 and SP6κ promoters and a control octamer-containing oligonucleotide (OCT). F indicates the free probe.

FIG. 2

The B29 promoter is induced by EBF in HeLa cells. (A) Luciferase activity assay results when a fos, a λ5, or a B29 promoter (+1 to −152) construct was transiently transfected into HeLa cells together with increasing amounts of EBF expression plasmid. The DNA content in each transfection was equalized by the addition of empty expression plasmid (cDNA3). The data represent four transfections from two independent experiments. (B) RT-PCR analysis of HeLa cells transiently transfected with EBF expression plasmid. The upper panel shows an ethidium bromide-stained agarose gel with the resulting amount of GADPH (glyceraldehyde-3-phosphate dehydrogenase) PCR products obtained from HeLa cells transfected with either an empty or an EBF-containing cDNA3 expression plasmid and from the human B-cell line Raji. The data show a serial dilution using 1, 1/5, 1/25 dilutions of the template cDNA after 25 cycles of PCR. The lower panel shows an autoradiogram from RT-PCR analysis indicating the amount of B29 transcripts in the same cDNA preparations after 25 cycles of PCR. The PCR products were blotted onto a nylon membrane and hybridized to an internal B29 oligonucleotide. The filters with PCR product from transfected HeLa cells were exposed for 24 h, while the corresponding filters for Raji cells were exposed for 4 h.

FIG. 3

Mutations in the three defined EBF binding sites impair the ability of the B29 promoter to bind EBF. (A) Schematic drawing indicating the mutations introduced into the B29 promoter by PCR using an oligonucleotide with inserted mutations. Lowercase letters indicate the mutations. (B) EMSA in which the binding of in vitro-translated recombinant EBF to the mb-1 promoter EBF binding site was competed for by the PCR-amplified (+1 to −152) wild-type B29 (B29), the mutated B29 (B29M), or the λ5 promoter. An actin PCR fragment served as a negative control. (C) EMSA using nuclear extracts from 230–238 pre-B cells and a consensus IKAROS binding site (3). The obtained band shift was competed for by an IKAROS consensus binding site, by the PCR-amplified wild-type (B29) or EBF mutant B29 (B29M) promoter, and by an octamer-containing oligonucleotide (OCT) as indicated. F indicates the free probe.

FIG. 4

The EBF binding sites are important for the function of the B29 promoter in pre-B cells. The resulting activities of wild-type (B29) and EBF-mutated (B29M) B29 promoter constructs relative to that of a basic luciferase vector (pGL3) after transient transfections into transformed mouse cells of the B-cell lineage are shown. The data are collected from four transfections and two independent experiments from the following cell lines: 230–238 pre-B cells, 18.81 pre-B cells, A20 mature B cells, S194 plasma cells, and J558 plasma cells.

FIG. 5

The B29 promoter interacts with EBF in 230–238 pre-B cells and has the ability to interact with multiple EBF homodimers simultaneously. (A) EMSA using an end-labeled truncated B29 promoter spanning nucleotides −105 to −152 and nuclear extracts from 230–238 pre-B cells. The obtained band shifts were competed for by the addition of the PCR-amplified full-length (+1 to −152) wild-type (B29) or EBF-mutated (B29M) B29 promoter; the mb-1 EBF, IKAROS consensus, or OCT protein binding site; and an actin PCR fragment as indicated. (B) EMSA using the B29 −105 to −152 probe and increasing amounts of either 230–238 pre-B-cell nuclear extracts or in vitro-translated recombinant EBF (rEBF). F indicates the free probe.

FIG. 6

EBF binding sites 2 and 3 are essential for the formation of the low-mobility complex. (A) Schematic drawing of the B29 promoter fragments that were used as binding sites for EBF in the EMSA. Introduced mutations are indicated by lowercase letters. (B) EMSA analysis with increasing amounts (0.5, 2, and 4 μl) of recombinant EBF and 20,000 cpm of the indicated end-labeled B29 promoter variants as binding sites. The band indicated by an asterisk was not competed for by an mb-1 promoter EBF binding site. F indicates free probe.

FIG. 7

EBF binding sites 2 and 3 are important for high-affinity binding and full functional activity of the B29 promoter. (A) Results of an EMSA in which the binding of 0.5 μl of recombinant in vitro-translated EBF to an excess of the mb-1 promoter EBF binding site was competed for by increasing amounts (50 and 100 times molar excess) of wild-type (B29) or EBF-mutated full-length B29 promoters (B29M1, B29M2, B29M3, M1-3, and B29M). The autoradiogram is cut to show only the EBF-DNA complex and not the free probe present in all lanes. (B) Luciferase activity resulting from DEAE-mediated transient transfection of luciferase reporter constructs controlled by wild-type (B29) or EBF site-mutated B29 promoters (M1, M2, M3, and M1-3) into 18–81 pre-B cells. Data represent the results of four experiments. (C) Resulting induction of activity of 250 ng of B29 promoter-controlled luciferase reporter gene in the presence of 400 ng of EBF expression plasmid after Lipofectin-mediated transient transfections into HeLa cells. The DNA content in each transfection was normalized with empty expression plasmid (cDNA3), and the data are taken from three independent transfections. Induction was calculated based on the activity of the different promoter constructs when transfected together with 400 ng of empty expression plasmid.