To determine the influence of LXRs and PU.1 on each other’s DNA binding profiles, LXR binding in the absence or presence of PU.1 was compared in PU1−/− and tamoxifen-treated PUER cells, and conversely, PU.1 binding was assessed in wild type and in LXRα/β double-knockout bone marrow-derived macrophages. Quantitative ChIP analysis for LXRβ in PU.1−/− vs. tamoxifen-treated PUER cells demonstrated that LXRβ recruitment to sites with vicinal PU.1 binding (within 100 bp) was PU.1-dependent (Figure 5F). This was not the case for LXRβ binding sites that were not in close proximity to PU.1 (Figure 5F), indicating that PU.1 determines LXR binding in a spatially restricted manner. In concert with these findings, macrophage-specific LXRβ- bound regions displayed a significant PU.1-dependent gain in H3K4me1 signal comparing H3K4me1 ChIP-Seq profiles in PU.1−/− vs. tamoxifen-treated PUER cells (Figure 5E). Conversely, the PU.1 cistrome and H3K4me1 patterns around macrophage-specific LXRβ-bound sites were not significantly altered in LXRα/β double-knockout macrophages (Figure 5G and 5H), suggesting that LXRs do not contribute to defining the PU.1 cistrome or to establishing the H3K4me1 pattern at these regions.
