To directly test if the binding of PU.1 to B lineage-specific sites is dependent on E2A, we retrovirally transduced E2A–deficient CLP/pre-pro B cells derived from the bone marrow of E2A−/− mice (Ikawa et al., 2004) either with a tamoxifen-inducible E2A–estrogen receptor ligand binding domain fusion protein (E47-ER), or the analogous construct coding for a deletion mutant of E47 that retains the bHLH DNA binding and dimerization domain but lacks both activation domains (bHLH-ER) (Sayegh et al., 2003). Upon activation of the fulllength E47-ER fusion protein with tamoxifen for 6 hours, PU.1 binding increased at 3752 sites > 4× relative to the bHLH-ER control, as exemplified by the IgK 3’ enhancer (Figure 3C), and these sites were strongly co-enriched for an E2A motif (Figure 3D and Figure S3A). In contrast, the PU.1 cistrome in these cells (44609 peaks total) was not significantly altered by expression of the E2A deletion mutant lacking transcriptional activation domains (Figure 3C).
