Taken together, our findings support a model in which two tiers of transcription factors cooperate to activate cis-active regulatory elements required for the development and function of macrophages and B cells (Figure 6E). The first tier consists of relatively small sets of lineage-determining factors, such as PU.1, C/EBPs and E2A, that act in a collaborative manner to delineate large sets of potential cis-regulatory elements in a cell type-specific fashion (exemplified for macrophages in Figure 6E). The specific attributes of these factors that enable this role remain to be defined, but presumably include expression levels and the ability to engage nucleosome remodeling factors and ultimately histone modifying enzymes that generate an accessible ‘proto-enhancer’ structure. Consistent with this, wild type E2A induces PU.1 binding at B cell-specific genomic sites that contain closely spaced PU.1 and E2A motifs, while a mutant form of E2A that is competent to bind to DNA but lacks transcriptional activation function does not (Figure 3C). This result highlights an important role for transactivation domains in enabling combinatorial transcription factor binding to chromatinized DNA at enhancer-like elements, in addition to their known roles in transcription initiation.
