Positive and negative transcriptional regulatory elements in the early H4 histone gene of the sea urchin,Strongylocentrotus purpuratus

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.