FIG. 4. Modulated expression of E2A-repressed genes in an inducible E2A reconstitution system.

A, E2A^−/− pre-B-cells were infected with E47R-IRES-GFP retrovirus (left plot), and GFP⁺ cells were sorted and cultured (right plot). B, tamoxifen treatment of E47R reconstituted B-cells restores inducible E2A DNA binding activity. Nuclear extracts from E2A⁺/⁺, E2A^−/−, and E47R reconstituted pre-B-cells were incubated with labeled μE5 oligonucleotide probe in the presence (even-numbered lanes) or absence (odd-numbered lanes) of a pan-E2A monoclonal antibody (YAE). Specific DNA binding is indicated by the arrows, and the supershifted band is shown by arrowheads. E2A DNA binding activity was detected in extracts from E2A⁺/⁺ (lanes 1 and 2) and E47R reconstituted cells treated with 1 μm tamoxifen (lanes 7 and 8) but not E2A^−/− (lanes 3 and 4) or 0.1% Me₂SO-treated E47R-reconstituted cells (lanes 5 and 6). C, RT-PCR analysis of total RNA isolated from pre-B-cell lines with either functional E2A (E2A^+/+), deleted E2A (E2A^−/−), or E2A^−/− cell line reconstituted with E47R fusion protein. Reconstituted cells were treated with either 0.1% Me₂SO (E47R(−)) or 1 μm tamoxifen in Me₂SO (E47R(+)) for 5 h to activate the E47R protein. Samples are presented as a series of serial 3-fold dilutions. D, tamoxifen treatment in the absence of E47R does not suppress gene expression. RT-PCR analysis of total RNA from E2A^−/− pre-B-cells mock-treated with 0.1% Me₂SO (−) or treated with 1 μm tamoxifen in Me₂SO (+) for 5 h. Each sample is presented as a series of 3-fold serial dilutions.