Although PU.1 binding sites are significantly correlated with expression of vicinal genes, (Figure 1E and 1F), an unbiased analysis of 71 sites in resting macrophages and B cells using a transient reporter gene assay revealed that a relatively small fraction (15%) conferred constitutive enhancer activity (Figure S6A). Given the finding that PU.1 participates in establishing the macrophage-specific LXR cistrome, it is possible that many of these sites might participate in defining the context-and signal-dependent transcriptional repertoire in each cell type. In support of this concept, regions containing both LXR and PU.1 binding sites exhibit significant sequence conservation in vertebrates, consistent with their potential importance as functional signal-responsive regulatory modules (Figure S5D). Functional analysis of representative LXR-PU.1 co-bound genomic regions vicinal to LXR target genes (as defined by their responsiveness to the synthetic LXR ligand GW3965 in primary macrophages, Figure S6B) by transient reporter gene analysis in RAW264.7 cells, demonstrated that 10 of 11 genomic regions tested exhibited ligand-dependent enhancer activity (Figure 6A). Furthermore, induction of LXR target genes in response to the LXR agonist GW3965 in PUER cells was markedly PU.1-dependent (Figure 6B).
