NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.
We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter.
Dugast
C
C
Laboratoire de Biologie Moléculaire Eucaryote, CNRS UMR 5099, 118 route de Narbonne, 31062 Toulouse, France.
Weber
M J
MJ
D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics*; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D019556:COS Cells; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018398:Homeodomain Proteins; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D050809:POU Domain Factors; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D014407:Tumor Cells, Cultured
D023081:CCAAT-Binding Factor; D018398:Homeodomain Proteins; D050809:POU Domain Factors; D014157:Transcription Factors; C085076:transcription factor Brn-2; D001142:Aromatic-L-Amino-Acid Decarboxylases