Transcription factor PU.1 controls transcription start site positioning and alternative TLR4 promoter usage

Human and mouse show markedly different sensitivities toward bacterial endotoxins, and recent evidence suggests that a species-specific regulation of the lipopolysaccharide receptor Toll-like receptor 4 (Tlr4) may contribute to this phenomenon. To gain further insight into mechanisms of Tlr4 regulation, we conducted a detailed in vivo and in vitro study of the murine Tlr4 gene, including analysis of transcription start site location, transcription factor occupancy, and activities of its proximal regulatory sequences. Our analyses identified a PU.1-dependent myeloid promoter, which is conserved between humans and mouse. We also identified an additional, distal promoter, located approximately 200 bp upstream of the myeloid-specific promoter, which is a functional target of E-box binding factors. In contrast to humans, where non-myeloid cells utilize both promoters, the distal Tlr4 promoter initiates all Tlr4 transcripts in murine non-myeloid cells, indicating that species-specific differences in TLR4 mRNA regulation may primarily exist in non-myeloid cell types. Interestingly, PU.1 null murine myeloid progenitor cells predominantly use the distal promoter, and the conditional induction of PU.1 expression in these cells leads to the rapid switch of transcription initiation to the proximal myeloid promoter. This indicates a direct role for PU.1 in determining the transcriptional start site and in recruiting the basal transcription machinery to myeloid promoters.