Expression of the thimet oligopeptidase gene is regulated by positively and negatively acting elements

Thimet oligopeptidase (TOP) is a thiol-dependent metallopeptidase, which ca n cleave and thereby modulate the activity of many neuropeptides. The enzym e is active in many endocrine tissues, including testis, brain, and pituita ry, In rat, the richest source of TOP is the testes, with a specific activi ty fivefold that of brain. The mechanism whereby rat TOP expression is regu lated at the transcriptional level has been examined by reporter gene assay and electromobility shift assays after isolation of 1020 bp of upstream se quence. Computer analysis predicts a number of potential transcription fact or-binding sites, which were examined by deletion analysis and DNA-binding studies. The promoter or its deletion fragments were fused to luciferase re porter gene vectors and introduced into GH3 pituitary, COS-1 kidney, MAT-Lu prostate, and GC-2spd(ts) spermatid cells. Two regions of the promoter hav e been identified: a positively acting region (-901/-219) and a strong nega tively acting region (-219/-102), Concomitantly, potential transcription fa ctors interacting with the cis-acting elements of the promoter were studied by gel electromobility shift assays. This work has identified a number of transcription factor-binding sites. However, no differences in the binding behavior in the various cell lines was observed.