Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes - A possible role for the DNA-binding protein Pur alpha

Nicotinic acetylcholine receptors constitute a multigene family (alpha 2-alpha 9, beta 2-beta 4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta 4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis reyealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Pur alpha. Electrophoretic mobility shift assays indicated that Pur alpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Pur alpha monoclonal antibody revealed the presence of Pur alpha, or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Pur alpha and the 19-bp element is critical for proper expression of the beta 4 subunit gene.