Analysis of the Negative Transcriptional Regulatory Element in the Angiotensin-Converting Enzyme Gene

We have characterized the sequence requirements and the protein binding properties of the previously identified transcriptional negative element present in the rabbit angiotensin-converting enzyme (ACE) gene. DNase footprinting experiments revealed that within the negative element (−715 to −610) several regions interact with proteins present in the nuclear extracts of ACE-expressing and -nonexpressing cell lines. Transfection analysis using the heterologous β-actin promoter and mutated negative elements demonstrated that the SP1 site, the collagen-silencer-like sequence, and the inverted repeat elements are dispensable for their functioning. Deletion of the region between −692 to −668 , however, completely eliminated the activity of the negative element, and mutation of the synapsin-silencer-like sequence present within this region vastly reduced it. This region (−692 to −668) by itself, when present in two copies, could effectively repress the activity of the β-actin promoter. The same point mutations in the silencer element that destroyed its action on the β-actin promoter greatly increased the transcriptional efficiency of the native ACE promoter. Electrophoretic mobility shift assay using the −692 to −668 ACE silencer sequence demonstrated the formation of a DNA/protein complex. UV cross-linking of the components of this complex revealed the presence of one prominent protein of approximately 21.5 kDa. This protein may be responsible for mediating the transcriptional-repressing activity of the ACE negative element. Homology between the ACE silencer and neuronal silencer consensus sequence, together with the promoter- and tissue-independent function of the the ACE silencer, suggests this element may bind a member of a large family of common negative regulatory transcription factors.