NEGATIVE REGULATORY ELEMENTS UPSTREAM OF A NOVEL EXON OF THE NEURONALNICOTINIC ACETYLCHOLINE-RECEPTOR ALPHA-2 SUBUNIT GENE

The expression of the nicotinic acetylcholine receptor a2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mecha nism underlying this regulation, we have investigated the structural a nd regulatory properties of the 5' sequence of this gene. A strategy b ased on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was fou nd almost-equal-to 3kb upstream from the first coding exon, and multip le transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nucl ear proteins, suggesting that the 1 kb flanking region contains at lea st a portion of the promoter of the gene. We have analysed the negativ e regulatory elements present within this region and found that a sile ncer region located between nucleotide - 144 and + 76 is active in fib roblasts as well as in neurons. This silencer is composed of six tande m repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member o f the Oct family. Moreover these motifs were found to act as a silence r only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested.