Involvement of PU.1 in the transcriptional regulation of TNF-α

PU.1 is a myeloid- and lymphoid-specific transcription factor that serves many important roles in the development and specific gene regulation of hematopoietic lineages. Mast cells (MC) and dendritic cells (DC) express PU.1 at low and high levels, respectively. Previously, we found that enforced expression of PU.1 in MC resulted in acquisition of DC-like characteristics, including repression of several IgE-mediated responses due to reduced expression of IgE-signaling related molecules. In contrast, PU.1 overexpression in MC up-regulated TNF-α production in response to IgE- and LPS-stimulation suggesting that PU.1 positively regulates TNF-α expression. However, the role of PU.1 in the expression of TNF-α is largely unknown. In the present study, the effects of PU.1 on the TNF-α promoter in mouse bone marrow-derived (BM) MC and DC were studied. Real-time PCR, ELISA, and chromatin immunoprecipitation assays indicated that the kinetics and magnitude of TNF-α expression levels following LPS- or IgE-stimulation are related to the amount of PU.1 binding to the promoter. In brief, higher and delayed up-regulation of TNF-α promoter function was observed in DC, whereas there were lower and rapid responses in MC. When PU.1-overexpressing retrovirus vector was introduced into MC, the amount of PU.1 recruited to the TNF-α promoter markedly increased. The knockdown of PU.1 in BMDC by siRNA resulted in a reduction of TNF-α protein produced from LPS-stimulated BMDC. These observations indicate that PU.1 transactivates the TNF-α promoter and that the amount of PU.1 binding on the promoter is associated with promoter activity.