Cooperative dimerization of the POU domain protein Brn-2 on a new motif activates the neuronal promoter of the human aromatic L-amino acid decarboxylase gene.
The neuronal promoter of the human aromatic L-amino acid decarboxylase (AADC) gene contains a perfectly palindromic element (TB) that conforms to the structure of a POU domain protein binding site of the MORE+2 type. The TB motif (located at nts -900/-872 relative to the neuronal cap site) bears striking similarities with the dimeric Pit-1 binding site from growth hormone gene promoter (GH-1), and it enhanced the activity of the minimal tk promoter in transfected SK-N-BE neuroblastoma cells. In transfected COS-7 cells, the expression of a 3xTB-tk-luc was stimulated up to 11-fold by the overexpressed Brn-2 protein. In AADC gene neuronal promoter, we previously characterized a bipartite regulatory element (ONF for octamer-like/NF-Y, nts -86/-57) that binds Brn-2 and NF-Y proteins in a cooperative manner. We now show that both TB and ONF sites participate in the activation of the neuronal promoter by Brn-2. EMSA experiments showed that the recombinant Brn-2 POU domain dimerized on the TB element in a cooperative manner. By site directed mutagenesis of the POU domain of Brn-2, the dimerization interface on the TB element was localized to the hydrophobic pocket of the POU specific domain and the C-terminal part of the POU homeodomain.
Dugast-Darzacq
Claire
C
Laboratoire de Biologie Moléculaire Eucaryote, CNRS UMR 5099/IFR 109, 118 route de Narbonne, 31062, Cedex, Toulouse, France. weber@ibcg.biotoul.fr
Egloff
Sylvain
S
Weber
Michel J
MJ
D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001859:Bone Neoplasms; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018398:Homeodomain Proteins; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009447:Neuroblastoma; D009838:Oligodeoxyribonucleotides; D050809:POU Domain Factors; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection
D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; D050809:POU Domain Factors; D014157:Transcription Factors; C085076:transcription factor Brn-2; D001142:Aromatic-L-Amino-Acid Decarboxylases