Phox2a regulates the expression of the human alpha3 nAChR gene by two mechanisms : DNA-dependent and DNA independent

Phox2a is a paired-like homeodomain transcription factor that participates in the specification of the autonomic nervous system. It is also involved in the transcriptional control of the noradrenergic neurotransmitter phenotype as it regulates the gene expression of the tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH), and norepinephrine transporter (NET). Here we show that Phox2a is capable of transactivating the human a3 nicotinic acetylcholine receptor (nAChR) subunit gene promoter by both DNA-dependent and DNA-independent mechanisms. Computer-assisted analysis of the human a3 gene promoter has revealed the presence of three putative consesus binding site (ATTA) for homeodomain-containing proteins, localised 0.6 Kb upstream the ATG codon, one within an Alu sequence and two immediately downstream of it. By means of EMSAs we demonstrate that Phox2a binds to the two ATTA sites downstream the Alu sequence. However, cotransfection experiments showed that the 60 bp a3 minimal promoter remains fully responsive to Phox2a even though it does not contain any putative cis-acting element for homedomain proteins. Both in vitro (DNA-pull down) and in vivo (ChIP) experiments confirmed the presence of Phox2a in the transcriptional complex that assembles on the a3 proximal promoter. As the a3 subunit is expressed in every terminally differentiated ganglionic cell (where it participates as a ligand-binding subunit in the formation of the ganglionic-type nicotinic receptor), it represents the first example of a "pan-autonomic" gene whose expression is regulated by Phox2a proteins.