To identify cis-acting elements involved with the expression of the rat carboxypeptidase-E (CPE) gene, constructs containing various regions of the 5′-flanking region of the CPE gene attached to the luciferase reporter gene were transiently expressed in cell lines derived from pituitary (AtT-20 and GH4C1), liver (SK-HEP-1), and kidney (HEK293 and COS1). Regions of the CPE gene spanning the major transcription initiation site (−12 to 47) are sufficient for low levels of transcription. Activity is enhanced 3- to 15-fold by sequences present between −12 and −395 in all cell lines examined. Sequences between −395 and −3081 influenced transcription activity up to 5-fold in some, but not all, cell lines. There was no correlation between the transcription activities of the various constructs and the level of endogenous CPE mRNA in the cell lines, indicating that the tissue-specific elements responsible for the large variations in endogenous CPE mRNA levels are not present within −3081 to 47. The region between −395 and 45 was examined in greater detail using transient expression assays and DNase-I protection analysis. Transcription activity is enhanced in GH4C1 and HEK293 cells by sequence present between −12 and −84; this region contains a potential GC box, which binds factors present in GH4C1 nuclear extracts. Other regions between −340 and 80 that bind proteins in the GH4C1 nuclear extracts include the major transcription initiation site, which has homology to the initiator sequence; the pituitary-specific transcription initiation sites (−101 and −105); and sequences with homology to NF-1, Pan-1, simian virus-40 enhancer core, and AP-2-binding sites. Taken together, these results suggest that basal expression of the CPE gene from its major transcription initiation site, which does not contain an up-stream TATA box, is primarily under the control of an initiator-like element together with an upstream GC box.
ASJC Scopus subject areas
- Molecular Biology