EXPRESSION OF THE CARBOXYPEPTIDASE-E GENE - CHARACTERIZATION OF THE INITIATOR-BINDING PROTEINS

Several of the genes for enzymes involved in peptide hormone processin g, such as carboxypeptidase E (CPE), do not contain a TATA box. The re gion surrounding the major transcription initiation site of the CPE ge ne has sequence homology with the 'initiator' (Inr) elements of the TA TA-less terminal deoxynucleotidyltransferase (TdT) gene, and the adeno virus major late (AdML) and other promoters. To investigate the promot er region of the CPE gene, GH(4)C(1) cells were transiently transfecte d with constructs containing the luciferase reporter gene attached to various portions of the rat CPE gene (-395 to +45). Positive regulator elements were detected in positions -84 to -12 and +30 to +47. Howeve r, the Inr-like element of the CPE gene (-12 to +20) produced detectab le luciferase activity in the absence of upstream and downstream seque nces. This region of the CPE gene was much more active when expressed in the normal (sense) orientation than when expressed in the antisense orientation. A mutation within the consensus sequence between CPE and other Inr elements was much less active than the wild-type sequence. Interestingly, deletion of the Inr and surrounding sequences produced a large increase in the transcription from upstream sites, suggesting that proteins which bind at, or near, the Inr sequence suppress transc ription from other sites. To characterize GH(4)C(1) nuclear proteins w hich bind to the CPE gene, Southwestern blotting, UV cross-linking, an d gel shift analyses were performed. The Southwestern analysis showed that the CPE and AdML Inr sequences labeled several proteins of simila r sizes which are distinct from the transcription factor USF; this fac tor has been previously reported to bind to the AdML Inr sequence. A C PE Inr-binding protein co-purifies with an AdML Inr-binding protein on a CPE Inr affinity column. Gel shift assays showed that with some bin ding conditions, the proteins that bind to the CPE sequence also bind to the TdT and AdML Inr elements. Taken together, these results indica te that the -12 to +20 region of the CPE gene has the properties of an Inr element which binds some, but not all, of the factors which bind to other Inr elements.