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 Bm-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. (C) 2001 Elsevier Science B.V. All rights reserved.