To test whether such PU. 1 dependence is a feature of other transcriptional programs defining macrophage-specific outputs, we assessed PU.1 dependence for TLR4 signaling responses in PUER cells. TLR4 ligation induces high magnitude changes in the expression of hundreds of genes in macrophages involved in innate immune responses through the activation of numerous signal-dependent transcription factors, including NF-κB and interferon regulatory factors (Lee and Kim, 2007). Transcriptome analysis of primary macrophages treated for 6 h with the specific TLR4 agonist Kdo2 lipid A (KLA) identified 735 genes induced more than 3-fold. PU.1 and C/EBPβ binding in resting macrophages occurred in the vicinity of 641 and 583 of these genes, respectively, and prior to KLA stimulation, these two factors co-localized, alone or together, with 537 of the 654 H3K4me1-marked regions associated with these genes (Figure 6C). Quantitative transcript analysis of 20 of these genes in the PUER system indicated that 14 required induction of PU.1 binding for qualitative or substantial quantitative responses to KLA (Figure 6D). Full responses of the remainder of these genes in untreated PUER cells indicated that the TLR4 signaling pathway is functional prior to activation of PU.1.
