Gene expression of the alpha subunit of a human translation initiation factor eIF-2, has been shown to be regulated during the mitogenic activation of quiescent T cells. Previous work has revealed that eIF- 2alpha is a single copy housekeeping gene with multiple start sites. Its promoter region is GC rich with five hypersensitive sites identified. Within one of these hypersensitive sites, a palindromic element was identified, which binds a transcription factor, alpha-PAL. Mutations of this element have been shown to affect eIF-2alpha promoter activity in a luciferase reporter gene construct transduced into NIH 3T3, 293 and COS cells. Downstream of the transcription start sites, an Inr (initiator) sequence has been identified that was oriented to allow transcription in the opposite direction to the sense transcript. The presence of this """"""""antisense"""""""" transcript has been demonstrated by RT-PCR in Go T cells, the level of which becomes reduced upon mitogenic stimulation of the cells. We have continued the analysis of the promoter region of eIF-2alpha. Region -560 to +1147 was cloned into a luciferase reporter gene construct in either the sense or antisense (opposite orientation) direction and transfected into either NIH 3T3, 293, COS or Jurkat cells. Analysis of luciferase activity showed weak activity for the sense constructs and strong activity for the antisense constructs. Deletion mutational analysis from either the 5' or 3' ends of either construct revealed that besides the alpha-PAL sites and Inr regions, there are other potential regulatory cis acting regions. We have identified an upstream activator region that is important to both the sense and antisense promoter activities, and a repressor region for the sense promoter within the region containing the Inr, corresponding to the antisense activity. By footprint analysis, we have identified an AP1 site with can be supershifted with c-fos and JunD antibodies. Also by footprint analysis with nuclear extract from activated T cells, we have identified a downstream region that binds nuclear factors. Gel-shift analysis with a variety of nuclear extracts from a variety of cells reveals binding activity which can be competed specifically with cold probe, but not with mutant probe. This region, however, when mutated reduced the antisense activity by only 20%. Deletion of the region surrounding the IRS sequence in the sense constructs increased its activity by 2.5 fold.
