Characterization of a novel stimulation-dependent secondary promoter in the lymphotoxin alpha gene (51.12)

Abstract

Abstract The lymphotoxin alpha (LTA) downstream region (DSR, +1 to +459) between the transcription and translation start sites contains polymorphisms that impact expression, yet the importance of the DSR to transcriptional regulation has not been examined. Similar regions in other genes have been shown to contain numerous regulatory elements and secondary promoter sites. We found that the LTA DSR is required for full activity of the LTA promoter in T and B cells as deletion of this region leads to a ≥55% reduction in luciferase expression under basal and stimulated conditions. Further, expression levels driven by the DSR alone were found to be comparable to that of the LTA regulatory region (-915 to +459) in basal and stimulated B and T cells. We hypothesized that the DSR contained a secondary promoter site that might consist of a putative Sp1 and/or TFII-I [initiator (Inr)-like element] binding sites located near the 3' end. Mutation of these sites resulted in significant reductions of DSR-mediated expression. ChIP assays showed that Sp1 and TFII-I, as well as RNA pol II, bind to this region of LTA in vivo. 5'RLM-RACE assays revealed LTA transcripts initiating from the Inr-like element when T cells were stimulated with human recombinant (hr) LT-α, hrTGF-β1 and hrKGF, but not when stimulated with PMA/ionomycin or left unstimulated. All transcripts were novel utilizing five novel start sites. These data show a complex stimulation-dependent pattern of LTA transcriptional regulation.