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 beta-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 beta-actin promoter. The same point mutations in the silencer element that destroyed its action on the beta-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.
Kessler
S P
SP
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195, USA.
Goraya
T Y
TY
Sen
G C
GC
D000818:Animals; D018983:DNA Footprinting; D003851:Deoxyribonucleases / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D003851:Deoxyribonucleases; D007703:Peptidyl-Dipeptidase A