The observation that PU. 1 binding occurred in the vicinity of motifs bound by lineage-restricted transcription factors prompted us to ask whether PU.1 binding to these sites was dependent on the presence of the respective factors. We addressed this question in B lineage progenitors devoid of both E2A and EBF (E2A−/−), EBF only (EBF−/−), or control cells that express both factors (Rag1−/−), which are arrested at successive stages in B cell development (Dias et al., 2008; Ikawa et al., 2004; Lin and Grosschedl, 1995; Mombaerts et al., 1992)(Figure 1A). ChIP-Seq for PU.1 in each of these cell populations yielded 44609, 36908 and 17210 peaks in E2A−/−, EBF−/− and Rag1−/− progenitors, respectively (FDR < 0.1%, Table S1). Surprisingly, despite the apparent loss in total number of PU.1 peaks comparing one developmental stage to the next, each successive stage was characterized by 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
