A distal enhancer is required for TNF gene transcription in human macrophages in response to TLR3 stimulation

Macrophages play a critical role in inflammatory responses during infections. These cells are activated by infections through stimulation of TLRs expressed on their cell surface and produce pro-inflammatory cytokines, including TNF. However, distal enhancers that regulate TNF gene transcription in human macrophages have not been investigated. This study used an unbiased genomic approach to identify six candidate enhancers in human primary alveolar macrophages within a 131 kb region from the transcription start site (TSS) of the TNF gene, covering 13 genes. Of these candidate enhancers, five showed enhancer activity, with three targeting the TNF gene and two targeting neighboring genes. Deletion of the distal TNF E-16 enhancer led to a 73% reduction in TNF gene transcription in response to poly (I:C) stimulation in the THP-1 human leukemia monocytic cell line. Additionally, deletion of the E-7.1/hHS-8 enhancer resulted in a 41% reduction in TNF mRNA, while deletion of the PE enhancer had a lesser effect, resulting in a 52% reduction in TNF gene transcription. Massively parallel reporter assays (MPRA) indicated that the transcription factor AP-1 and EGR1-binding sites at the distal TNF E-16 enhancer were crucial in mediating enhancer activity. This study shows that both distal and proximal enhancers work together to fully transcribe the TNF gene in human macrophages in response to TLR ligand poly (I:C) stimulation.