IDENTIFICATION OF POSITIVE AND NEGATIVE TRANSCRIPTIONAL REGULATORY ELEMENTS OF THE RABBIT ANGIOTENSIN-CONVERTING ENZYME GENE

The two tissue-specific mRNAs encoding the isozymes of rabbit angioten sin-converting enzyme (ACE) are generated from the same gene by altern ative choice of two transcription initiation sites 5.7 kb apart. In th e current study, we have characterized the regulatory sites controllin g the transcription of the larger pulmonary isozyme mRNA. For this pur pose, reporter genes driven by varying lengths of upstream region of t he ACE gene were transfected into ACE-producing cells. Our results dem onstrated that the transcription of this gene is primarily driven by p ositive elements within the first 274 bp DNA upstream of the transcrip tion initiation site. The reporter gene driven by this region was expr essed in two ACE-producing cells but not in two ACE-non-producing cell s thereby establishing its tissue specificity. Our experiments also re vealed the existence of a strong negative element located between -692 and -610 positions. This element suppressed the expression of the rep orter gene in a dose-dependent and position and orientation-independen t fashion thus suggesting that it is a true silencer element. It could also repress the expression of a reporter gene driven by the heterolo gous strong promoter of the P-actin gene. The repressing effects of th e negative element could be partially overcome by cotransfecting the i solated negative element along with the reporter gene containing the n egative element. This result was possibly due to the functional remova l of a limiting trans-acting factor which binds to this element. Elect rophoretic mobility shift assays revealed that the negative element ca n form several complexes with proteins present in the nuclear extract of an ACE-producing cell line. At least part of the negative element i s strongly conserved in the upstream regions of the human and mouse AC E genes.