U4B snRNA gene enhancer activity requires functional octamer and SPH motifs

Expression of the chicken U4B small nuclear RNA (snRNA) gene is stimulated by a transcriptional enhancer located approximately 190-227 base pairs upstream of the transcription start site. This enhancer is composed of at least two functional motifs: an octamer (binding site for Oct-1) and an SPH motif. We now report that these two motifs functionally cooperate to stimulate U4B snRNA gene expression, and both are required for the formation of a stable transcription complex. Expression in frog oocytes of 24 different point mutant constructions indicates that the functional SPH motif is at least 15 base pairs in length. It is a recognition site for a sequence specific DNA-binding protein, termed SBF, purified from chicken embryonic nuclear extracts. The ability of the mutant SPH motif constructions to be recognized by SBF in vitro correlates with their transcriptional activities, suggesting that SBF mediates the stimulatory effect of the U4B SPH motif. These results are similar to our recent findings on the chicken U1 gene enhancer, which also contains adjacent binding sites for Oct-1 and SBF. These studies, together with evolutionary considerations and sequence comparisons among snRNA gene enhancers, suggest that cooperativity between octamer and SPH motifs could be a widely-employed mechanism for generating vertebrate snRNA gene enhancer activity.