We next evaluated the consequences of PU.1 activity on C/EBPβ binding in PU.1−/−cells stably expressing a tamoxifen-responsive PU. 1-estrogen receptor ligand binding domain fusion protein (PUER) (Walsh et al., 2002). In this inducible system low levels of PU.1 activity and DNA binding are observed by ChIP-Seq in the absence of tamoxifen (8144 peaks total, exemplified in Figure 3E, PUER-PU.1–0h). This low level PU.1 activity was associated with partial recovery of C/EBPβ binding in the vicinity of some PU.1 binding sites (Figure 3E and Figure S3H, PUER- C/EBPβ-0h). Tamoxifen treatment induced PU.1 binding to a total of 37909 genomic regions after 1 h and resulted in a gain of 1710 C/EBPβ binding sites (>4× tags relative to the 0 h time point), restoring a large number of macrophage-specific PU.1 binding sites. The induced C/EBPβ binding regions were strongly enriched for a PU.1 motif (Figure 3F and Figure S3B) and were co-bound by PU.1 at over 75% of sites. Intriguingly, 57% of PU.1 binding sites observed in the absence of tamoxifen were co-bound by C/EBPβ, implying C/EBP and related factors play an important role in directing PU.1 localization when effective PU.1 concentrations are low.
