Abstract
The early H4 (EH4) histone gene of the sea urchin, Strongylocentrotus purpuratus, is shown to contain at least five positive-responding sequence elements and one negative-responding site which control the level of in vitro transcription in an embryonic nuclear extract. The positive acting elements are: 1) the UHF-1 region, located between -133 and -102 (the site of a strong footprint, due at least in part to the binding of an 85 kD protein factor termed UHF-1); 2) the H4 specific element (H4SE), situated between -62 and -39; 3) a sequence corresponding to a TATA box between -33 and -26 (TAACAATA); 4) the transcriptional initiation site; and 5) an internal sequence element found between +19 and +50. Deletion of, or base changes in, the UHF-1, H4SE, initiation, or internal sequence sites resulted in significant decreases in transcription. Base substitutions in the TATA-like sequence had much less effect, resulting in no more than a 2-fold decrease in transcription, and there was no evidence that alternative initiation sites are utilized in the mutant templates. The negative element (termed the UHF-3 site) is contained within a footprinted region between nucleotides -75 and -56. Base substitutions in this area result in templates that were transcribed at a level 1.2-2.0-fold higher than the wild-type gene. Transcription levels of double UHF-1/H4SE and UHF-1/INR mutants were those expected from additive effects of the individual mutations and there was no suggestion of synergism.