ENHANCER CONTAINING UNUSUAL GC BOX-LIKE SEQUENCES ON THE HUMAN CHOLINE-ACETYLTRANSFERASE GENE

The minimum requirement of an enhancer for the human choline acetyltra nsferase (ChAT) gene was analyzed by mutagenesis and protein-DNA inter action studies. A series of deletion and site-specific mutants were ex pressed transiently in a rat cholinergic neuroblastoma cell, NS20Y. Th e results revealed that the distal region between -970 and -941 base p airs (bp) from the transcription start site is essential for efficient transcription of the human ChAT gene. Two GC box-like sequences were located within this region, and they were shown to bind purified Sp1 t ranscription factor by footprinting and gel retardation analyses. This sequence, however, has an orientational effect and is located approxi mately 1000 bp from the transcription start site, unusual for the conv entional GC box sequence. Furthermore, the sequence between these GC b ox-like sequences is shown to bind another novel trans-acting factor b y gel retardation assay and to be necessary for efficient enhancer act ivity. On the other hand, gel retardation assays using a nuclear extra ct from Drosophila SL2 cells suggested that non-Spl trans-acting facto rs could also participate in the enhancing activity of this element. T aken together, the results demonstrate that two GC box-like sequences and another trans-acting factor-binding sequence located between -970 and -941 bp act coordinately and are essential for efficient transcrip tion of this low expressed human ChAT gene.