the appearance of PU.1 sites at new genomic positions that were not occupied by PU.1 in the previous progenitor stage. Using the PU.1 sites identified in E2A−/− cells as control, de novo motif analysis of the 3760, 2479, and 9504 PU.1 sites exhibiting 3× more tags in the EBF−/−, Rag1−/−, and mature B cells, respectively, revealed enrichment for distinct motifs for transcription factors expressed at respective stages of B cell development (Figure 3A and Table S3), demonstrating that PU.1 binding is dependent on the activity of other lineage-determining transcription factors. For example, co-enriched E2A motifs were gained comparing the PU.1 cistrome in EBF−/− cells to that in E2A−/− cells (Figure 3A). Furthermore, gained sites were primarily associated with genomic regions corresponding to B cell-specific PU.1 binding sites in mature splenic B cells, as exemplified by the reshaping of the PU.1 binding site pattern comparing CLP/pre-pro B (EBF−/− ) to pro-B cells (Rag1−/−) (red data points in Figure 3B).
